Compositions of vaccines and adjuvants and methods for the treatment of urinary tract infections

ABSTRACT

This invention describes novel adjuvant compositions and formulations with excellent stability at refrigerated and room temperatures and up to and about 37° C. that can be produced at remarkably low costs. This invention describes novel vaccine compositions and formulations to treat and prevent urinary tract infections caused by gram-negative bacteria including  Escherichia coli  and multi-drug resistant  E. coli . This invention also describes methods of administration of said novel vaccine compositions and formulations and methods of treatment to prevent and treat urinary tract infections caused by gram-negative bacteria including  E. coli  and multi-drug resistant  E. coli.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation in part of U.S. application Ser. No.14/494,001, which claims benefit of U.S. Provisional Application No.61/882,498 filed on Sep. 25, 2013, the contents of which areincorporated in its entirety by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention provides novel adjuvant compositions and formulations withexcellent stability at refrigerated and room temperatures, and also upto about 37° C., that can be produced at remarkably low costs. Thesenovel adjuvant compositions and formulations are used in vaccines andexhibit superior properties of enhancing immune responses to antigenswhile causing less severe injection site and systemic reactions. Theinvention also describes novel vaccine compositions and formulations totreat and prevent urinary tract infections caused by gram-negativebacteria including Escherichia coli and multi-drug resistant E. coli.The invention also provides methods of administration of said novelvaccine formulations and methods of treatment to prevent and treaturinary tract infections caused by gram-negative bacteria including E.coli and multi-drug resistant E. coli.

2. Description of the Related Art

In the United States (US) and other countries most populations areprotected from numerous infectious diseases by the use of vaccines.Vaccines protect people from infectious diseases such as diphtheria,tetanus, pertussis, hepatitis, influenza, and polio to name a few.Society relies on the protections afforded by vaccines, which haverendered most of these infectious diseases only a part of history forthe citizens of the US. In fact, in the US, the Centers for DiseaseControl (CDC) administer the Vaccines for Children program whichprovided free vaccinations for approximately 40 million children in2010. Approximately 70% of these children are enrolled in Medicaid. Toprevent outbreaks of disease and reduce the costs of treating theseinfectious diseases, the US has made vaccination a national priorityindependent of economic status. Low-cost vaccines are desperatelyrequired and these vaccines are a national priority now and in theforeseeable future.

Given the importance of vaccines, the need to continually develop newand improved vaccines to improve the health of our population is clear.Even more critical is the need to provide lower cost vaccines to assistwith reducing the skyrocketing costs of the US healthcare system. Anational priority is to lower the costs of the US healthcare system.

Contributing to these difficulties, even in the US, is compliance withvaccine storage requirements. A study conducted by the Office of theInspector General at the Department of Health and Human Services andreported in 2012 (OE1-04-10-00430) found that providers participating inthe Vaccines for Children Program of the CDC: 1.) exposed vaccines totemperatures outside their approved temperature ranges; 2.) storedvaccines in refrigerators and freezers at temperatures outside theirapproved temperature ranges; and 3.) had expired vaccines stored withnon-expired vaccines.

Another issue with vaccines is that vaccines can have a short shelf lifeand are prone to expire prior to use.

In addition, the study described above conducted by the Office of theInspector General found that 16 of 46 US healthcare providers ofVaccines for Children program had expired vaccines stored with unexpiredvaccines. On average, these expired vaccines had been expired for about6 months. For example, it was reported that as of Jul. 1, 2010 40million unused doses of swine flu vaccine that cost about $260 millionto produce had just expired and were being destroyed. Vaccineexpirations result in significant economic losses each year in the US.

Adjuvants enhance the immune responses to antigens of vaccines. Of the34 vaccines provided under the Vaccines for Children Programadministered by the CDC in the US, 20 contain adjuvants. Of these 20vaccines with adjuvants, 19 of these vaccines contain alum adjuvants and1 vaccine contains monophosphoryl lipid A adsorbed to alum (GSK's MPL)as the adjuvant.

Despite industry wide attempts at developing new adjuvants, currentlyonly alum and GSK's MPL are used in approved vaccines in the US.Numerous adjuvant development failures have occurred in the US, but theneed for new and effective adjuvants remains high.

GlaxoSmithKline's (GSK) Cervarix vaccine containing3′-O-desacyl-4′-monophosphoryl lipid A adsorbed to alum (GSK's MPL) waslicensed in the US for the prevention of cervical cancer caused by humanpapillomavirus. Because the starting material to produce MPL is isolatedfrom Salmonella minnesota, the final product is a dynamic, complexmixture of hexa-, penta-, and tetraacyl analogues; each of theseanalogues differ in biological activity. As a result, the mixture of3′-O-desacyl-4′-monophosphoryl lipid A presents manufacturing, testing,and use challenges that greatly contribute to the expense and supplyissues with the vaccine.

In addition to storage problems, vaccine injections are often painful tothe recipient. Redness, swelling, itching and tenderness at injectionsites may occur after administration of a vaccine. The PrescribingInformation of GSK's Cervarix Vaccine containing MPL and alum adjuvantslists local adverse events that may include pain, redness, and swelling.Local pain that prevented activities of daily life was reported inapproximately 8 percent of subjects receiving either GSK's Cervarixvaccine or the adjuvant alum alone. Systemic adverse reactions observedafter administration of vaccines containing MPL and alum adjuvantsinclude headache, fatigue, fever, rash, myalgia, arthralgia, urticaria,and gastrointestinal symptoms including nausea, vomiting, diarrhea,and/or abdominal pain.

Furthermore, as described in the “Clinical Review of HumanPapillomavirus Bivalent (Types 16 and 18) vaccine [GSK's CervarixVaccine], Recombinant, Biologics License Application EfficacySupplement” four studies reported local adverse events including localpain preventing movement in approximately 16 percent of subjects.Swelling was also reported at greater than 50 mm in approximately 3percent of subjects. The same four studies reported systemic severeadverse events in 2.4 to 7.8 percent for arthralgia, fatigue,gastrointestinal, headache, and myalgia.

The severity of the injection site reactions and systemic reactions aresignificant requiring medical treatment involving narcotic use, IVhydration, or other physician implemented treatments and loss of workfrom preventing daily activity due to diarrhea, myalgia, fatigue,headache, and vomiting.

The Advisory Committee on Immunization Practices establishesrecommendations for the National Strategy for Pandemic Influenza. Thisstrategy includes the need to “provide pandemic vaccine to all UScitizens within 6 months of a pandemic declaration: pandemic vaccine(600 million doses) [National Strategy for Pandemic Influenza (November2005) and HHS Pandemic Influenza Plan (November 2005)” and requires theuse of adjuvants to attempt to move toward this seemingly unapproachablevaccination target. Since there is no approved adjuvant for a generalflu vaccine in the US, the US national vaccine stockpile was without analternative and purchased the MF59 adjuvant from Novartis for about $500million. The MF59 adjuvant was recently discontinued in a clinical studyof Fluad Paediatric due to “high vaccine reactogenicity observed inchildren 9 through 12 years of age, the protocol of study V7P29 wasamended to exclude children less than 9 years of age.” The evidencesupports that during a national pandemic declaration a significantnumber of severe reactions will occur due to the use of the MF59adjuvant and require additional medical care.

A synthetic analogue of monophosphoryl lipid A was introduced by AvantiPolar Lipids (Alabaster, Ala., USA) in around 2004 time period. AvantiPolar Lipids named this synthetic analogue phosphorylated hexaacyldisaccharide “PHAD”, alternatively known as “GLA”. PHAD supplied byAvanti Polar Lipids is provided as a single compound, shown in FIG. 1,of approximately 98% purity with a molecular weight of 1763 Daltons.PHAD's purity is in stark contrast to GSK's MPL isolated from Salmonellaminnesota that, as described above, exists as a dynamic, complexmixture. Unlike GSK's MPL, PHAD's manufacturing process, supply, use,and stability can be closely monitored and controlled as a purecompound.

Whether or not a specific adjuvant or combination of adjuvants willenhance an immune response toward each specific antigen isunpredictable. For example, GSK's Cervarix vaccine contains bothmonophosphoryl lipid A and alum, because this combination is superior toalum alone (Giannini et al. Vaccine, 2006, 24, p. 5937-5949). A similarincrease in efficacy was observed with a vaccine that used a recombinanthepatitis B surface antigen. (Vaccine, 1998, 16(7), p. 708-714). Anotherexample demonstrating the variability of antigen—adjuvant combinationsin producing an immune response for a specific antigen is shown in Table6 of U.S. Pat. No. 6,889,885. These inventors demonstrated that theQS-21 adjuvant and, separately, the alum plus monophosphoryl lipid Aadjuvant combination generated greater antibody responses to a 74kDprotein than alum or monophosphoryl lipid A alone. Furthermore, in 2009Derek T. O'Hagan and Ennio De Gregorio of Novartis Vaccines published areview about the development of adjuvants. (Drug Discovery Today,14(11/12), June 2009, p. 541-551) They reported that alum is arelatively weak adjuvant for certain proteins or antigens and newadjuvants are still required.

In 2004 the Infectious Disease Society of America (IDSA) forewarned apending crisis of increasing antibiotic resistant bacteria throughoutthe world with no new antibiotics on the horizon to combat thisoccurrence. In 2009 the IDSA identified that bacterial infections nowoccur that are resistant to all current antibiotics, and that the mostalarming antibiotic resistant bacteria are gram-negative bacteriaincluding E. coli. In 2010, the IDSA stated that despite efforts by manyprivate, public, and government laboratories, research had not producedany new alternatives to treat antibiotic resistant bacteria and a globalcommitment was now required. IDSA's urgency is supported by scientistsat GlaxoSmithKline who predicted it would be greater than ten to fifteenyears prior to the launch of any new antibiotics for the treatment ofgram-negative bacterial infections (Payne et al. Nature Reviews DrugDiscovery. 2007, 6, p. 29-40).

Their prediction was based upon the failure of 34 companies thatattempted to develop new antibiotics. A consensus among the scientificcommunity is emerging that the US urgently needs new treatments forbacterial infections. Adam L. Hersh and colleagues reported a surveywith 562 infectious disease physicians responding across the US in thejournal of Clinical Infectious Disease in 2012 (Hersh et al. CID. 2012.54(11), 1677-8) that 63% of these physicians had treated patients withbacterial infections resistant to all known antibiotics within the lastyear. These data emphasize the need for new treatments for bacterialinfections. The failure in the art to identify new therapeuticalternatives to prevent and treat gram-negative bacterial infections iswell documented.

Moreover, at least five vaccines under development to prevent or treatStaphylococcus aureus infections have recently been discontinued. Theseinclude StaphVAX, Veronate, Aurexis, Aurograb, and V710. The failure toidentify new vaccines to prevent and treat bacterial infections is welldocumented.

Urinary tract infections (UTIs) are one of the most prevalent infectiousdiseases worldwide and the number one infectious disease suffered bywomen in the US. Symptoms of UTIs include dysuria (painful urination),urgency to urinate, and suprapubic pain. Acute uncomplicated UTIs occurin an estimated 7 to 11 million women in the US each year. Over half ofall adult women will suffer from one or more UTIs in their lifetime with25-44% of women experiencing a recurrent UTI. In fact, approximately1,000,000 women and men in the US experience three or more UTI episodesper year. Moreover, recurrence often occurs within 30 to 90 days ofinfection despite appropriate antibiotic treatment and apparentclearance of the initial infection from the urine.

Despite recent progress in the epidemiology and pathogenesis of UTI,there have been no recent major improvements in our ability to actuallyprevent or treat these infections. The 25 to 44% of women with UTI whoexperience recurrent infections require additional treatment, additionalcosts, and in some cases extensive urological evaluation to prevent moresevere complications from arising. Thus, safe and effective vaccinesthat have the potential to improve patient convenience and decreasecosts are of considerable interest to patients, providers, and healthcare organizations. In the recurrent UTI population, antimicrobialresistance is of great concern since treatment options are diminishing.There is, therefore, an urgent need to develop new approaches to UTIprevention and treatment that depend less on the use of antimicrobials.

UTIs are most commonly caused by uropathogenic Escherichia coli (UPEC),which can be responsible for up to 85% of community-acquired UTIs. Acritical pathogenic cascade by which UPEC evade host defenses andrapidly expand in numbers in the urinary tract to cause disease has beenuncovered. This work supports the clinical need for a UTI vaccine.

FimH plays a significant role in several stages of the pathogenesiscascade, which makes it a prime vaccine target. UPEC strains that lackthe FimH adhesin are unable to effectively colonize the bladder. Avaccine against FimH will activate host defenses to recognize and clearUPEC at all stages of infection, even when protected in IBCs orintracellular reservoirs.

A FimCH vaccine with MF59 as the adjuvant containing squalene wasjointly invented by scientists at MedImmune, Inc. and the laboratory ofProfessor Scott Hultgren (U.S. Pat. No. 6,500,434; incorporated hereinin its entirety). The FimH protein and FimC protein exist as anon-covalent protein complex, FimCH. FimC stabilizes FimH and antibodiesare produced against both proteins, however, only antibodies to FimHhave been shown to reduce E. coli colonization of bladders in animals.The use of FimCH as an antigen in a vaccine is therefore limited by therequirement of an effective adjuvant.

The FimCH vaccine with the MF59 adjuvant containing squalene (anoil-in-water emulsion) elicited an immune response during Phase 1clinical trials (United States Patent Application 20030138449;incorporated herein in its entirety.). Phase 2 clinical trials wereconducted in two distinct populations again with MF59 containingsqualene, but women did not produce relevant IgG titers to FimH ineither of these trials. The development of MedImmune's FimCH vaccinewith the MF59 adjuvant was discontinued because of these disappointingresults. MF59 with squalene has a history of causing severe localinjection site and systemic reactions when used with certain antigens.During these Phase 2 clinical trials, women experienced severe injectionsite reactions and severe systemic reactions. Because of this failure, avaccine for the treatment or prevention of UTI does not exist in the US.

An ongoing need exists for a vaccine to prevent and treat UTI. Thefailure of MedImmune and others to develop a UTI vaccine evidences thedifficulties of developing new vaccines for bacterial infections.MedImmune demonstrated that alum does not sufficiently enhance theimmune response to FimCH. MedImmune had no clear alternatives ofadjuvants to pair with the FimCH antigen.

Accordingly, there is an urgent need for vaccines, and adjuvants used toenhance the immune response to antigens in vaccines. There is a need forvaccines and adjuvants for vaccines that have extended stability withoutsacrificing efficacy. In particular, there is an urgent and widelyrecognized need in for more room temperature stable vaccines andadjuvants. In addition, it would be desirable to have vaccines,adjuvants and compositions that are stable at temperature above roomtemperature.

In addition there is a need for adjuvants and pharmaceuticalcompositions that produce less severe injection site and systemicreactions.

There is a need for new vaccines to prevent and treat bacterialinfections, and for vaccines for the prevention and treatment of UTIs inparticular.

It would be desirable to have vaccines, and adjuvants used to enhancethe immune response of antigens in vaccines, with increased shelf-livesthat can be produced in a cost effective manner. Such vaccines andadjuvants would significantly lower healthcare costs in the US,particularly if they can be stored at room temperature or greaterwithout negatively affecting their stability.

It would be desirable to have adjuvants and vaccines that produceminimal injection site and systemic reactions. It would be desirable tohave formulations with as few as excipients as possible.

It would be desirable to have a vaccine, and adjuvant for a vaccine thatenhances the immune response treat bacterial infections. It would bedesirable to have a vaccine, and adjuvant for a vaccine that enhancesthe immune response to Escherichia coli to patients with UTI.

BRIEF SUMMARY OF THE INVENTION

The present invention addresses many problems of prior art adjuvants,vaccines and pharmaceutical compositions described herein. The presentinvention provides novel liquid adjuvant compositions and formulationswhich provides many unexpected and advantageous properties unknown inthe art of adjuvant and pharmaceutical compositions.

In one aspect, liquid adjuvant compositions and formulations thatexhibit room temperature stability for about more than 6 months and upto about 37° C. for about 60 or more days is provided. The novel liquidadjuvant compositions and formulations can be stored at refrigerated orroom temperature conditions facilitating its shelf life during shippingand storage and lowering its delivery costs.

The adjuvant formulations of the invention described herein address manycurrent obstacles in vaccine administration by enabling a low cost andunexpectedly and remarkably stable adjuvant formulation which enhancesan immune response to E. coli antigen with less severe injection siteand systemic reactions.

The data described herein demonstrate that the adjuvant formulations ofthe invention enhance the immune response to other antigens includingbacterial and viral antigens.

The invention described herein contributes to reducing this problem bytreating urinary tract infections caused by gram-negative bacteriaincluding E. coli.

In one aspect, a novel adjuvant composition with remarkable stability at2° C. to 8° C. and room temperature up to about 37° C. is disclosed.

In one aspect of the invention, a composition comprising onesynthetically produced adjuvant PHAD and a buffer selected from thegroup consisting of citrate, succinate, and phosphate at about 25 mM toabout 50 mM, preferably 28 mM to about 50 mM, and most preferably 30 mMto about 50 mM. These novel PHAD compositions are preferably aqueousbuffered suspensions. The composition can be used in a variety of waysin the vaccine and pharmaceutical context. The composition, withpreferably no additional components, significantly improves thestability of PHAD in suspension and achieves exceptional stability atroom temperature and up to and at about 37° C. The compositions alsoexhibit excellent stability at refrigerated temperature as well. Thisrepresents a significant advancement in adjuvant and pharmaceuticaltechnology by providing an efficient and economical PHAD compositionthat does not require refrigeration for long term stability.

In another aspect of the invention, novel adjuvant formulations as anaqueous buffered suspension are provided. In one embodiment the adjuvantformulations include one synthetically produced adjuvant PHAD, a bufferselected from the group consisting of citrate, succinate, and phosphateat about 10 mM to about 50 mM, preferably about 25 mM to about 50 mM,more preferably 28 mM to about 50 mM, and most preferably 30 mM to about50 mM, and preferably one synthetically produced phosphatidylcholine.When a phosphatidylcholine is added, the preferred buffer concentrationscan be expanded to about 10 mM to about 50 mM and achieve the remarkablestability described herein. These novel adjuvant formulations arepreferably aqueous buffered suspensions. The adjuvant formulations haveexcellent long-term stability when stored at refrigerated and roomtemperatures and excellent stability up to and at about 37° C. Theseformulations can be produced at remarkably low costs.

The novel adjuvant formulations described herein do not requirelyophilization, or equivalent process for room temperature stability orstability up to and at about 37° C. The adjuvant formulations include aspecific buffer and optionally and preferably one or more syntheticallyproduced phosphatidylcholines selected from the group consisting ofDMPC, DPPC, DSPC, DOPC, and POPC, preferably DPPC, and one syntheticallyproduced adjuvant, PHAD, in a molar ratio of about 1:1 to 40:1(phosphatidylcholine:PHAD), preferably about 1:1 to 20:1(phosphatidylcholine:PHAD), more preferably about 2:1 to 5:1(phosphatidylcholine:PHAD), and most preferably about 2:1 to 5:1(DPPC:PHAD).

One of the most valuable aspects of the invention is that the adjuvantformulations include only a single adjuvant PHAD in citrate, succinateor phosphate buffers at specified concentrations as described herein,and preferably a single phosphatidylcholine. No other ingredients arerequired to produce the unexpected long-term stability at roomtemperature. Further the adjuvant formulation can be produced at lowcost. In this regard, the long-term stability of these adjuvantformulations at room temperature is remarkable and is achieved withoutthe use of cholesterol, phosphatidylglycerol, phosphatidylethanolamine,monoacylglycerol, lyoprotectants, and metabolizable oil. Conventionalprior art adjuvants do not achieve stability without the use of one ormore of these ingredients.

Another aspect of this invention is adjuvant formulations that do notneed two or more phosphatidylcholines or the addition of aphosphatidylglycerol. As shown in the examples, two or morephosphatidylcholines or one or more phosphatidylglycerols can be addedto these formulations, but preferably it is not needed to achieve theremarkable long-term stability demonstrated herein.

While not bound by theory, expansion of the preferred bufferconcentrations of citrate, succinate, or phosphate buffer to about 10 mMto about 50 mM to achieve the remarkable stability of the inventiondescribed herein is believed to be due to addition of preferredexcipients, preferably phosphatidylcholine, at the defined molar ratioof the preferred aspect of the invention of phosphatidylcholine to PHADdescribed herein. More preferably, the preferred buffer concentrationsare about 25 mM to about 50 mM, even more preferably 28 mM to about 50mM, and most preferably 30 mM to about 50 mM. Preferably the pH is in arange of about 4.0 to about 7.5, preferably about 4.5 to about 6.5, morepreferably about 5.0 to 6.0.

As shown in the examples, this exceptional stability at room temperatureis not present when formulated in water, acetate buffer, PBS, or citrateor phosphate buffers at or greater than 100 mM. Instead the stability isproduced by citrate, succinate, or phosphate at concentrations of about10 mM to about 50 mM, but preferably about 25 mM to about 50 mM, morepreferably 28 mM to about 50 mM, and most preferably 30 mM to about 50mM.

Another embodiment of the invention includes a non-ionic surfactant,preferably polysorbate 80, to reduce the aggregation of particles of theinvention.

Removing the need for lyophilization is a significant advantage andunexpected breakthrough because many costly steps and risks have beeneliminated. Another aspect of this invention is that these adjuvantformulations are superior at enhancing an immune response to an antigenwhile causing significantly less severe injection site and systemicreactions during administration compared to the prior art. Anotheraspect of this invention is adjuvant formulations substantially free ofmetabolizable oils, including squalene, and substantially free ofcholesterol. In the art, it is widely considered that cholesterol isnecessary for adjuvant formulations or liposomes to function. Thecurrent invention produces all its benefits as described herein withoutrequiring cholesterol.

Therefore, the advantages of the adjuvant formulations andpharmaceutical compositions described herein include: room temperaturestability as an aqueous buffered suspension for at least 6 months,and/or stability up to and at about 37° C. for about 60 or more days;less severe injection site and systemic reactions per administrationwhile enhancing immune responses to antigens; and, lower cost ofproduction or manufacturing with fewer materials or components and lowerconcentration of the materials or components. The inventive adjuvantformulations provide these three combined major benefits not previouslyachieved by adjuvants that are synthetic analogues of MLA or MPL asalternatives to alum-based adjuvants.

In another aspect of the invention novel vaccine compositions containingthe novel adjuvant formulations for use to treat and prevent urinarytract infections caused by gram-negative bacteria including Escherichiacoli and multi-drug resistant E. coli are provided. Methods ofadministration of said novel vaccine compositions and methods oftreatment to prevent and treat urinary tract infections caused bygram-negative bacteria including E. coli and multi-drug resistant E.coli are also provided.

In another aspect of the invention methods of inducing the production ofantibodies against FimH in a human with recurrent urinary tractinfections are provided.

Another aspect of the invention is vaccine compositions that induce theproduction of antibodies against FimH in a human with recurrent urinarytract infections.

In another aspect of the invention, a vaccine kit comprising the PHADcompositions or formulations or vaccine compositions, along withadministration directions and instructions for storage are provided. Theinstructions provide for the exposure of the PHAD compositions orformulations at room temperature and up to and about 37° C. Theseinstructions describing storage, shipping, and exposure temperatures maybe approved by a government regulatory authority including the US FDA orEuropean Medicines Agency. Preferably, one or more of the kit componentsis the PHAD compositions or formulations in a syringe.

Another aspect of the invention is that the PHAD composition andformulations and vaccine compositions are sterile compositions andsterile pharmaceutical compositions, more preferably the sterile PHADcompositions and formulations are contained in a sterile syringe.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the chemical structure of one salt of PHAD

FIG. 2 shows the chemical structure of DPPC

FIG. 3 is a graph illustrating the indirect ELISA of FimCH and Q133Kusing IgG anti-FimH.

FIG. 4 is a graph illustrating the potency assay analyzing FimCH andQ133K.

FIG. 5 is a graph illustrating the evaluation of small moleculeinhibitors in the potency assay. Two small molecules,4-methylumbelliferyl-α-D-mannopyranoside (UFMP) andmethyl-α-D-mannopyranoside (MDMP) inhibit mannose binding to FimH.

FIG. 6 is a representative chromatogram of the FimCH drug substancesample by CEX-HPLC.

FIG. 7 is a graph illustrating the protection from E. coli Infectionfollowing FimCH/PHAD immunization of mice.

FIG. 8 is an example chromatogram of DPPC and PHAD by HPLC.

DETAILED DESCRIPTION OF THE INVENTION Definitions

“About” in reference to PHAD quantity or buffer concentration (unlessdefined) means plus and minus 10% of the listed quantity.

“About 25° C.” refers to temperatures at 20° C. to 30° C.

“About 37° C.” refers to temperatures at 34° C. to 40° C.

“About 50 mM” refers to the buffer concentrations described herein meansbetween 50 mM and less than 100 mM. As shown in the examples, 100 mM orgreater of the specified buffers is not as effective in enablinglong-term room temperature stability. The upper end of bufferconcentrations is typically evaluated at two-fold increments. Withreference to about 50 mM, the specified buffer concentrations of theinvention are preferably less than 90 mM, preferably less than 80 mM,more preferably less than 70 mM, and most preferably less than 60 mM.

“About 10 mM” refers to the buffer concentrations described herein meansbetween 6 mM and 10 mM.

“Acceptable carrier” refers to a carrier that is not deleterious to theother ingredients of the composition and is not deleterious to materialto which it is to be applied.

“Adjuvant” refers to an agent that, when present in an effective amount,increases the antigenic response; a substance enhancing the immuneresponse to an antigen; or an agent that stimulates antibody productionto an antigen. Numerous naming conventions or terminologies exist in theart. Without reference to a specific naming convention, the adjuvantcompositions as described herein may simply be referred to as adjuvantformulations or adjuvant preparations.

“Administration” refers to any means of providing a compound orcomposition to a subject.

“Colloid” refers to one or more chemicals, compounds, or substancesmicroscopically dispersed throughout an aqueous buffered solution oranother substance. The adjuvant formulations described herein can alsobe described as colloid. One example of a colloidal dispersion isFungizone, which consists of Amphotericin B-sodium desoxycholate forparenteral administration.

“Critical micelle concentration” refers to the concentration ofsurfactant(s) above which micelles form and all additional surfactantsadded to the system go to micelles.

“DLPC” refers to 1,2-dilauroyl-sn-glycero-3-phosphocholine.

“DMPC” refers to 1,2-dimyristoyl-sn-glycero-3-phosphocholine.

“DOPC” refers to 1,2-dioleoyl-sn-glycero-3-phosphocholine.

“DPPC” refers to 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (molecularformula C₄₀H₈₀NO₈P (MW=734 Da) (the chemical structure is shown in FIG.2).

“DPPG” refers to 1,2-dipalmitoyl-sn-glycero-3-phospho-(1′-rac-glycerol).

“DSPC” refers to 1,2-distearoyl-sn-glycero-3-phosphocholine.

“Effective amount” refers to a sufficient amount of FimCH or truncatedFimH or other antigen in a vaccine composition that is administered to ahuman to elicit an immune response against FimH or other antigen, orsufficient amount of an adjuvant, preferably PHAD, to elicit anincreased immune response to antigen.

“Essentially free” in reference to materials, additives, chemicals, orexcipients means the materials, additives, chemicals, or excipients havenot been added to the composition or formulation of the invention,although some impurity level amounts may be present.

“Essentially free of severe injection site and systemic reactions” meanstwo percent or less of humans experience these severe injection site andsystemic reactions that are attributable to the adjuvant composition orformulation.

“Injection site reaction” refers to pain, tenderness, redness, and/orswelling at the site of administration or injection site.

“Invention” means at least some embodiments of the present invention;references to various feature(s) of the “invention” or “presentinvention” throughout this document do not mean that all claimedembodiments or methods include the referenced feature(s).

“Labeling” or “Label” refers to all labels and other written, printed,or graphic matter upon any article or any of its containers or wrappers,or accompanying such article and, therefore, includes any packageinserts or information sheets that accompany vaccine or adjuvantcompositions or formulations of the invention.

“Less severe injection site and systemic reactions” refers to lesssevere or Grade 3 injection site reactions and/or systemic reactions ascompared to commercial vaccine Cervarix as detailed herein and in itsproduct information documents and investigational vaccine adjuvantGLA-SE as described herein and in Treanor et al. (Vaccine 2013).

“Liposome” refers to generally vesicles that consist of a lipid bilayermembrane surrounding a hydrophilic core.

“Low cost” or “low costs” refers to a composition with the components ormaterials at the lowest concentrations sufficient to achieve the novelcharacteristics of the invention.

“Lyoprotectants” refers to materials, chemicals, or excipients primarilyused to protect materials from freezing damage or other impairmentduring manufacturing, storage and use or improving reconstitutionincluding enabling appropriate solvation prior to use, and also includesthese materials, chemicals, or excipients used to modify osmolality oradjust tonicity, and include but not limited to sorbitol, mannitol,mannose, erythritol, xylitol, glycerol, sucrose, dextrose, trehalose,maltose, lactose, and cellobiose.

“Metabolizable oil” refers primarily to squalene, or closely relatedanalogues of squalene as used as an adjuvant in vaccine formulations oradjuvant formulations, but also refers to medium-chain triglyceridesincluding Miglyol 810 and oils from vegetables, animals, or fish whenused in vaccine or adjuvant formulations as excipients, or to create anadjuvant effect, or to produce emulsions. Examples include grapeseedoil, soybean oil, coconut oil, olive oil, sunflower oil, corn oil, andshark liver oil.

“Micelle” refers to an aggregate of surfactant molecules dispersed in anaqueous buffered solution with the hydrophilic head regions in contactwith surrounding aqueous buffered solution, sequestering the hydrophobicsingle-tail regions in the center of the micelle.

“MLA” refers to monophosphoryl lipid A.

“MPL” refers to 3′-O-desacyl-4′-monophosphoryl lipid A.

“Pharmaceutically acceptable carrier” refers to a carrier that is notdeleterious to the other ingredients of the composition and is notdeleterious to the human or other animal recipient thereof. In thecontext of the other ingredients of the composition, “not deleterious”means that the carrier will not react with or degrade the otheringredients or otherwise interfere with their efficacy. Interferencewith the efficacy of an ingredient does not, however, refer to meredilution of the ingredient.

“PHAD” is a Toll-like receptor 4 agonist and refers to phosphorylatedhexaacyl disaccharide or pharmaceutically acceptable salts ofphosphorylated hexaacyl disaccharide. The structure of a preferred PHADsalt is shown in FIG. 1, which is available from Avanti Polar Lipids.PHAD, as used herein may be fully or partially synthetic ornon-synthetic, although fully synthetic is preferred.

“Pharmaceutical composition” refers to a composition given to a mammalintended to treat or prevent a disease, or in the case of a vaccinecomposition, to produce an immunogenic response that treats or preventsa disease, reduce symptoms, or provides some type of therapeuticbenefit, or in the case of an adjuvant composition, to enhance an immuneresponse to one or more antigens.

“Phosphate buffer” or “phosphate” refers to a phosphate buffer selectedfrom the following group: sodium phosphate dibasic, sodium phosphatemonobasic, potassium phosphate monobasic, and potassium phosphatedibasic, or some combination thereof. Preferably “phosphate” consists ofsodium phosphate dibasic, sodium phosphate monobasic, and potassiumphosphate monobasic. Unless otherwise noted, reference to phosphatebuffer specifically excludes ammonium phosphate.

“PBS” refers to phosphate-buffered saline of a general composition ofphosphate buffer (Na₂HPO₄ and/or KH₂PO₄), potassium chloride and sodiumchloride. A typical PBS composition is comprised of about 10 mMphosphate buffer (Na₂HPO₄ and/or KH₂PO₄), 2.7 mM potassium chloride and0.14 M sodium chloride, pH 7.4, at 25° C.

“Phosphate citrate buffer” refers to a phosphate buffer containingcitric acid and sodium phosphate where the pH is maintained bycitrate/citric acid and phosphate/hydrogen phosphate equilibrium. Thephosphate may include, for example Na₂HPO₄ and/or KH₂PO₄ and trisodiumcitrate may be used.

“Phosphatidylcholine” (alternatively referred to as “PC”) refers tolipids containing choline. Examples include, but not limited to, DMPC(1,2-dimyristoyl-sn-glycero-3-phosphocholine), DPPC(1,2-dipalmitoyl-sn-glycero-3-phosphocholine), DSPC(1,2-distearoyl-sn-glycero-3-phosphocholine), DOPC(1,2-dioleoyl-sn-glycero-3-phosphocholine), and POPC(1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine). Phosphatidylcholinesare available from Avanti Polar Lipids.

“Phosphatidylethanolamine” refers to lipids containing a phosphate groupattached to an ethanolamine, e.g.1,2-dioleoyl-sn-glycero-3-phosphoethanolamine.

“Phosphatidylglycerol” refers to lipids containing glycerol. Examplesinclude, but are not limited to,1,2-dimyristoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (DMPG),1,2-dipalmitoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (DPPG), and1,2-distearoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (DSPG).

“POPC” refers to 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine.

“Recurrent urinary tract infections” refers to a human has 3 to 4Urinary Tract Infections in approximately one year.

“Refrigerated” refers to a temperature range from 2° C. to 8° C.

“Room temperature” refers to a range of temperature from 19° C. (66° F.)to 25° C. (77° F.).

“Saline” refers to about 125 mM to about 155 mM of NaCl in bufferedaqueous solutions. For example, PBS generally contains 137 mM NaCl andTris buffered saline may contain 150 mM.

“Severe injection site reaction” refers to one or more of the following:pain requiring narcotic pain reliever or that prevents daily activity;tenderness causing significant discomfort at rest; redness of more than10 cm; and swelling of more than 10 cm or prevents daily activity.

“Severe systemic reaction” refers to one or more of the following:nausea/vomiting which prevents daily activity or requires outsubject IVhydration; diarrhea consisting of 6 or more watery stools or >800 gramswith 24 hours or requires outsubject IV hydration; headache consistingof significant use of narcotic pain reliever or prevents daily activity;fatigue consisting of significant or prevents daily activity; andmyalgia consisting of significant or prevents daily activity.

“Substantially free” in referring to cholesterol means that cholesterol,if present, is at 0.3 mM or less.

“Substantially free” in referring to monoacylglycerol means,monoacylglycerol, if present, is at 0.5 mM or less. An example ofmonoacylglycerol is monopalmitoyl glycerol.

“Substantially free” in referring to phosphatidylglycerol orphosphatidylethanolamine means these substances, if present, are at 0.1mM or less.

“Substantially free” in referring to lyoprotectants means thesesubstances, if present, are at a concentration of the composition orformulation of 0.5% or less.

“Substantially free of saline” means less than 30 mM NaCl in thecomposition or formulation of the invention.

“Systemic reactions” refers to nausea/vomiting, diarrhea, headache,fatigue, and/or myalgia.

“Succinate buffer” or “succinate” refers to disodium succinate or sodiumsuccinate dibasic. Potassium succinate may be used, but is lesspreferred.

“Stability” or “stable” in reference to adjuvants, actives, proteins,antigens, or drugs refers to the quality of the substance or product toremain acceptable for its intended use throughout a certain time periodbeginning from its date of manufacture while under the influence of suchvariables as temperature and/or humidity. The stability of a substanceis often demonstrated by analytical data (or other equivalent evidence).

“Trisodium citrate” refers to citrate buffers (also referred to as“citrate”) such as, for example, trisodium citrate dihydrate, sodiumcitrate, sodium citrate tribasic hydrate, or citric acid trisodium saltdihydrate as referred to by suppliers including Sigma-Aldrich and BDHChemicals. Potassium citrate, sodium citrate monobasic, and sodiumcitrate dibasic may be used, but they are less preferred. For example,the product imiglucerase for injection uses a combination of trisodiumcitrate and disodium hydrogen citrate. These types of combinations areacceptable. Citric acid, CAS 77-92-9, may be used to adjust the pH ofthe buffer, but it cannot substitute for the listed buffers herein.

“Truncated FimH” refers to the FimH protein truncated to include atleast about 25 to about 175 amino acid residues from the first 175 aminoacids of FimH. With reference to truncated FimH, the FimH proteintruncated to include preferably at least 9% of the FimH protein, morepreferably at least 30% of the FimH protein, and most preferably atleast 60% of the FimH protein.

“Urinary Tract Infections” refers to a medical diagnosis characterizedby 1 or more of the following signs and symptoms: irritative voidingsuch as frequency, urgency, and dysuria; gross hematuria; or elicitedsuprapubic tenderness upon examination; and/or 1 or more of thefollowing laboratory results: positive urine dipstick test from cleancatch or catheter urine specimen; microscopic urinalysis from cleancatch or catheter urine specimen (leukocytes, bacteria, and casts may bepresent); or urine culture from clean catch or catheter urine specimenfor E. coli at ≧10³ CFU/mL.

“Vaccine” or “vaccine composition” refers to a composition that improvesimmunity to a disease. The vaccine compositions are immunogeniccompositions that elicit immune responses and antibody production towardthe antigen of the composition.

EMBODIMENTS OF THE INVENTION

In one embodiment a pharmaceutical composition or pharmaceuticallyacceptable carrier is provided. The pharmaceutical compositions orpharmaceutically acceptable carriers are comprised of PHAD and a buffer(referred to as “PHAD composition” or “PHAD containing composition” or“PHAD containing composition and carriers”). The PHAD compositions ofthe invention are preferably aqueous buffered suspensions. The buffer isselected from the group consisting of citrate, succinate, and phosphateat about 25 mM to about 50 mM, more preferably 28 mM to about 50 mM, andmost preferably 30 mM to about 50 mM. Preferably the pH is in a range ofabout 4.0 to about 7.5, preferably about 4.5 to about 6.5, morepreferably about 5.0 to about 6.0. The pharmaceutical composition orcarrier with this combination (PHAD and the buffer) demonstratesexcellent stability as a base for pharmaceutical compositions andimproves the overall stability of PHAD compositions. Specifically thesePHAD containing compositions and carriers achieve stability at roomtemperature and up to about 37° C. These PHAD containing compositionsand carriers of the present invention also exhibit excellent long-termstability at refrigerated temperatures to room temperature.

The PHAD containing pharmaceutical compositions and pharmaceuticalcarriers (which as previously described include PHAD and a specificbuffer) may optionally include other ingredients typical to vaccines,adjuvant formulations, and other pharmaceutical compositions such asexcipients, modifiers, surfactants, and additives. For one example,phosphatidylcholines (as described in more detail below) may optionallybe added, alone or in combination with other lipid carriers. In oneembodiment naturally derived phosphatidylcholines from soy or egg orhydrogenated phosphatidylcholines from soy or egg or synthetic ornatural mixed acyl phosphatidylcholines may be added.

In a preferred embodiment, one or more vaccine antigens are added to thePHAD containing compositions to form a vaccine. The vaccine antigens maybe any antigen used in vaccines including, but not limited todiphtheria, tetanus, pertussis, poliomyelitis, hepatitis, and orantigenic preparations of the influenza virus. Preferably the vaccineantigen is a FimCH protein complex as described in detail below.

The PHAD containing compositions and carriers can be prepared at anyconcentration but are typically prepared at about 0.005 to about 1.0mg/ml of PHAD, preferably about 0.05 to 1.0 mg/ml of PHAD, but usuallynot more than about 2.5 mg/ml of PHAD.

The PHAD containing compositions may be administered to animals orhumans as an adjuvant of vaccines to preferably deliver about 10microgram of PHAD per dose to about 50 micrograms of PHAD per dose. Theexact dose may be modified according to the antigen used. Morepreferably about 20 micrograms of PHAD per dose to about 50 microgramsof PHAD per dose is administered. Even more preferably about 40micrograms of PHAD per dose to about 50 micrograms of PHAD per dose isadministered.

As shown herein, the unexpected stability of the PHAD containingcomposition at room temperature and up to 37° C. is absent whenformulated in water, acetate buffer, PBS, or citrate or phosphatebuffers at or greater than 100 mM. The PHAD compositions containcitrate, succinate, or phosphate buffer to produce the unexpectedstability, preferably about 25 mM to about 50 mM, more preferably 28 mMto about 50 mM, and most preferably 30 mM to about 50 mM.

More particularly, preferred buffers of the PHAD composition, theconcentrations of the buffers are selected from the group consisting ofabout 25 mM to about 50 mM; 25 mM to about 50 mM; about 30 mM to about50 mM; 28 mM to about 50 mM; 30 mM to about 50 mM, about 30 mM; about 40mM to about 50 mM; 40 mM to about 50 mM; about 40 mM; and about 50 mM.

In addition, as shown in the examples below, the use of PBS in the PHADcontaining compositions and formulations do not exhibit the novelcharacteristics of the invention, and in particularly the stabilitycharacteristics of the invention. In contrast, the use of citrate,succinate, and phosphate buffers as specifically defined herein enablesthe novel stability characteristics of the invention.

The PHAD compositions of the invention are preferably essentially freeof squalene.

The PHAD compositions of the invention are preferably essentially freeof metabolizable oil used as an adjuvant.

The PHAD compositions of the invention are preferably essentially freeof metabolizable oil.

The PHAD compositions of the invention are preferably aqueous bufferedsuspensions, and preferably these aqueous buffered suspensions haveparticle sizes less than 150 nm, even more preferably less than 130 nm,and preferably these PHAD compositions are not oil-in-water emulsions.

The PHAD compositions of the invention are preferably essentially freeof a second adjuvant including alum, squalene, QS21, MF59, Toll-likereceptor 9 agonists, and other adjuvants including squalene-basedadjuvants. Second adjuvants have the potential to generate more severelocal and systemic reactions in humans yet without the benefit offurther increasing an immune response to improve therapeutic outcomes.

The PHAD compositions preferably contain less than 5 mM of cholesterol,more preferably less than 1 mM of cholesterol, and even more preferablysubstantially free of cholesterol, and most preferably essentially freeof cholesterol.

Preferably, the PHAD compositions are substantially free ofphosphatidylglycerol, and more preferably are essentially free ofphosphatidylglycerol.

Preferably, the PHAD compositions are substantially free ofphosphatidylethanolamine, and more preferably essentially free ofphosphatidylethanolamine.

Preferably, the PHAD compositions are substantially free ofmonoacylglycerol, and more preferably essentially free ofmonoacylglycerol.

The PHAD compositions are preferably substantially free of saline,preferably contain less than 20 mM NaCl, and more preferably containless than 10 mM NaCl, and even more preferably essentially free of NaCl.

The PHAD compositions are preferably substantially free oflyoprotectants, and even more preferably essentially free oflyoprotectants.

The PHAD compositions do not require lyophilization, or equivalentprocess, to preserve the concentration of PHAD for shelf-life orstability. Therefore, the PHAD compositions are preferably notlyophilized or lyophilization does not occur; the PHAD compositions arepreferably not dried after preparation; the PHAD compositions preferablydo not require reconstitution from dried material with liquid or bufferafter preparation; and the PHAD compositions preferably remain as anaqueous buffered suspension after manufacturing prior to administration.

Preferably, the PHAD compositions are substantially free of cholesterol,phosphatidylglycerol, and phosphatidylethanolamine, and essentially freeof metabolizable oils and a second adjuvant.

More preferably, the PHAD compositions are essentially free ofcholesterol, phosphatidylglycerol, and phosphatidylethanolamine, andessentially free of metabolizable oils and a second adjuvant.

Preferably the PHAD compositions of the invention are substantially freeof materials or excipients as described herein that are not required toachieve the novel characteristics of the invention, but even morepreferably the PHAD compositions of the invention are essentially freeof materials or excipients as described herein that are not required toachieve the novel characteristics of the invention.

Preferably the PHAD compositions of the invention are substantially freeof one, two, three, or more of materials or excipients per compositionas described herein, and this limitation is not exclusive to only onematerial or excipient per composition.

Preferably the PHAD compositions of the invention are essentially freeof one, two, three, or more materials or excipients per composition asdescribed herein, and this limitation is not exclusive to only onematerial or excipient per composition.

Preferably, when a PHAD containing composition or formulation of theinvention is stored, shipped, held, or administered at room temperatureor up to about 37° C., it is most preferred to have been sterilefiltered or prepared using sterile techniques, more preferably thesterile PHAD compositions and formulations are contained in a sterilesyringe.

In another embodiment novel adjuvant formulations are provided. Theadjuvant formulations include one synthetically produced PHAD, a bufferselected from the group consisting of citrate, succinate, and phosphateat about 10 mM to about 50 mM, and preferably one synthetically producedphosphatidylcholine (referred to as “PHAD formulations” or “PHADcontaining adjuvant formulations” or “PHAD containing formulations”).The phosphatidylcholine and PHAD are present at a molar ratio of about 1(PC) to 1 (PHAD) to about 40 (PC) to 1 (PHAD), preferably about 2.5 (PC)to 1 (PHAD). Reference to PHAD formulations apply to the adjuvantformulations unless otherwise noted. These novel PHAD formulations arepreferably aqueous buffered suspensions. The adjuvant formulations haveexcellent long-term stability when stored at refrigerated temperaturesto room temperatures, and up to about 37° C. Additionally, the adjuvantformulations can be produced at low costs.

In a preferred embodiment, the adjuvant formulations include preferablyone synthetically produced phosphatidylcholine, preferably DPPC, and onesynthetically produced adjuvant PHAD, in a molar ratio of about 1:1 to40:1 (DPPC:PHAD), and citrate, succinate, or phosphate buffers at about10 mM to 50 mM, but preferably about 25 mM to 50 mM, more preferably 28mM to about 50 mM, and most preferably 30 mM to about 50 mM. Preferablythe pH is in a range of about 4.0 to about 7.5, preferably about 4.5 toabout 6.5, more preferably about 5.0 to about 6.0. Importantly, the PHADadjuvant formulations can be produced with only one adjuvant PHAD andpreferably one phosphatidylcholine, thereby enabling them to be producedat low cost. Preferably, the adjuvant formulations contain PHAD as thesole adjuvant, however additional adjuvants may be used. The long-termstability attribute of the invention further reduces costs of usingthese PHAD containing adjuvant formulations.

While not bound by theory, expansion of the preferred bufferconcentrations of citrate, succinate, or phosphate buffer to about 10 mMto about 50 mM in the PHAD formulations to achieve the remarkablestability of the invention described herein is believed to be due toaddition of a preferred excipient, preferably phosphatidylcholine, atthe defined molar ratio of the preferred aspect of the inventionphosphatidylcholine to PHAD described herein.

The PHAD containing adjuvant formulations of the invention arepreferably essentially free of squalene.

The PHAD containing adjuvant formulations of the invention arepreferably essentially free of metabolizable oil used as an adjuvant.

The PHAD containing adjuvant formulations of the invention arepreferably essentially free of metabolizable oils.

The PHAD containing adjuvant formulations of the invention arepreferably aqueous buffered suspensions, and preferably these aqueousbuffered suspensions have particle sizes less than 150 nm, even morepreferably less than 130 nm, and preferably these PHAD containingadjuvant formulations are not oil-in-water emulsions.

The PHAD containing adjuvant formulations of the invention arepreferably essentially free of a second adjuvant including alum,squalene, QS21, MF59, Toll-like receptor 9 agonists, and other adjuvantsincluding squalene based adjuvants. The PHAD formulations are preferablyessentially free of other or additional adjuvants because they have thepotential to generate more severe local and systemic reactions in humanswithout the benefit of further increasing an immune response orsubstantially improving therapeutic outcomes.

The PHAD containing adjuvant formulations preferably contain less than 5mM of cholesterol, more preferably less than 1 mM of cholesterol, evenmore preferably substantially free of cholesterol, and most preferablyessentially free of cholesterol.

Preferably, the PHAD containing adjuvant formulations are substantiallyfree of phosphatidylglycerol, and more preferably essentially free ofphosphatidylglycerol.

Preferably, the PHAD containing adjuvant formulations are substantiallyfree of phosphatidylethanolamine, and more preferably essentially freeof phosphatidylethanolamine.

Preferably, the PHAD containing adjuvant formulations are substantiallyfree of monoacylglycerol, and more preferably essentially free ofmonoacylglycerol.

The PHAD containing adjuvant formulations of the invention arepreferably substantially free of saline, preferably contain less than 20mM NaCl, and more preferably, the PHAD formulations contain less than 10mM NaCl, and even more preferably are essentially free of NaCl.

Preferably, the PHAD containing adjuvant formulations are substantiallyfree of lyoprotectants, and more preferably essentially free oflyoprotectants.

Preferably, the PHAD containing adjuvant formulations are substantiallyfree of cholesterol, phosphatidylglycerol, and phosphatidylethanolamine,and essentially free of metabolizable oils and a second adjuvant.

More preferably, the PHAD containing formulations are essentially freeof cholesterol, phosphatidylglycerol and phosphatidylethanolamine, andessentially free of metabolizable oils and a second adjuvant.

Preferably the PHAD containing formulations of the invention aresubstantially free of materials or excipients as described herein thatare not required to achieve the novel characteristics of the invention,but even more preferably the PHAD containing formulations of theinvention are essentially free of materials or excipients as describedherein that are not required to achieve the novel characteristics of theinvention.

Preferably the PHAD containing formulations of the invention aresubstantially free of one, two, three, or more of materials orexcipients per PHAD containing formulation as described herein, and thislimitation is not exclusive to only one material or excipient per PHADcontaining formulation.

Preferably the PHAD containing formulations of the invention areessentially free of one, two, three, or more materials or excipients perPHAD containing formulation described herein, and this limitation is notexclusive to only one material or excipient per PHAD containingformulation. In another aspect, vaccines, and methods of treating andpreventing disease with vaccines, are provided.

To prepare the vaccines, one or more vaccine antigens are added to thePHAD containing adjuvant formulations described above. The antigens maybe a FimCH protein complex as described herein or other antigensincluding, but not limited to antigens associated with diphtheria,tetanus, pertussis, poliomyelitis, hepatitis, and or antigenicpreparations of the influenza virus.

A vaccine prepared using the PHAD formulation does not requirelyophilization to preserve the concentration of PHAD for shelf-life orstability. Lyophilization is a dehydration process or freeze-dryingprocess primarily utilized to preserve materials. Equivalent processesexist to accomplish the same goal to preserve materials. A vaccine oradjuvant formulation that does not need lyophilization is an unexpectedand significant advantage in the preparation of vaccines and the PHADcompositions described herein. By eliminating the need forlyophilization, many costly steps are eliminated. First, thelyophilization step itself is eliminated, which not only removes acostly manufacturing step that must occur under well-monitored sterileconditions, but also eliminates the validation and review of thismanufacturing step. Next, the removal of the step represents an ongoingcost saving. For example, each time a lot is prepared a lyophilizationstep is saved. In addition, removing this step saves extra validationsteps when larger batches are prepared or the manufacturing procedure istransferred to another facility. Second, lyophilization requires that asterile vial of diluent be manufactured or procured, shipped, and storedwith the lyophilized product for reconstitution. Eliminating thereconstitution step removes the cost of this diluent vial and its supplychain management. Third, the reconstitution of adjuvant formulations cancompromise its sterility, necessitating its immediate use or waste ofthe product if not used in a pre-established amount of time. Fourth, thereconstitution process is prone to errors, so the manufacturer losescontrol of the exact concentration of the product that is ultimatelyadministered to a patient. The adjuvant compositions and formulationsdescribed herein have eliminated these four disadvantages by removingthe lyophilization requirement. Therefore, the PHAD containingformulations are preferably not lyophilized or lyophilization does notoccur; the PHAD containing formulations are preferably not dried afterpreparation; the PHAD containing formulations preferably do not requirereconstitution from dried material with liquid or buffer afterpreparation; and the PHAD containing formulations preferably remain asan aqueous buffered suspension after manufacturing prior toadministration.

A further remarkable advantage of the invention described herein is nowrealized. Without a need for lyophilization or equivalent process, thePHAD compositions or PHAD containing formulations of the invention canbe efficiently and cost effectively packaged into syringes immediatelyafter manufacturing. Preferably the compositions and formulations aresterile and preferably the syringes are sterile. These prefilledsyringes can be shipped, stored, delivered, or transferred atrefrigerated temperatures to room temperatures and up to about 37° C.This advantage provides the most efficient and cost effective means toget PHAD compositions and PHAD containing formulations to a site foradministration.

In another embodiment, a non-ionic surfactant is added to the adjuvantformulation. Preferably the non-ionic surfactant is polysorbate 80although others may be used. The non-ionic surfactant is typically addedat a concentration of about 0.001% to 1.0%, preferably 0.01% to 0.1%, tothe adjuvant composition. This addition can prevent slight aggregationor slight increase in mean particle size of the PHAD formulations whilebeing stored at room temperatures and up to about 37° C. Preferably,non-ionic surfactants are derived from polyethoxylated sorbitan andinclude but are not limited to polysorbate 20 and polysorbate 80.

Preferred adjuvant formulations comprise a specific buffer selected fromthe group consisting of citrate, succinate, and phosphate at about 25 mMto about 50 mM, more preferably 28 mM to about 50 mM, and mostpreferably 30 mM to about 50 mM, and preferably one syntheticallyproduced phosphatidylcholine selected from the group consisting of DMPC,DPPC, DSPC, DOPC, and POPC, preferably DPPC, and one syntheticallyproduced adjuvant, PHAD, in a molar ratio of about 1:1 to 40:1(phosphatidylcholine:PHAD), preferably about 1:1 to 20:1(phosphatidylcholine:PHAD), more preferably about 2:1 to 5:1(phosphatidylcholine:PHAD), and most preferably about 2:1 to 5:1(DPPC:PHAD). More preferably, citrate or succinate buffers are used inthe PHAD formulations at about 10 mM to about 50 mM, preferably at about25 mM to about 50 mM, more preferably 28 mM to about 50 mM, and mostpreferably 30 mM to about 50 mM.

As described herein referring to the concentrations of the preferredbuffers of the PHAD formulation, the concentrations of the buffers areselected from the group consisting of about 10 mM to about 50 mM; 15 mMto about 50 mM; 20 mM to about 50 mM; about 25 mM to about 50 mM; 25 mMto about 50 mM; about 30 mM to about 50 mM; 28 mM to about 50 mM; 30 mMto about 50 mM, about 30 mM; about 40 mM to about 50 mM; 40 mM to about50 mM; about 40 mM; and about 50 mM. The PHAD containing formulationsdescribed herein are preferably substantially free of saline, preferablycontain less than 20 mM NaCl, and more preferably, the PHAD compositionscontain less than 10 mM NaCl and even more preferably essentially freeof NaCl.

In addition, as shown in the examples below, the use of PBS in the PHADcontaining compositions and formulations do not exhibit the novelcharacteristics of the invention, and in particularly the stabilitycharacteristics of the invention. In contrast, the phosphate buffers asspecifically defined herein do enable the novel characteristics of theinvention.

The PHAD formulations are preferably prepared by selecting a singlephosphatidylcholine prepared synthetically with high purity. However,naturally derived phosphatidylcholines from soy or egg or hydrogenatedphosphatidylcholines from soy or egg or synthetic or natural mixed acylphosphatidylcholines may also be used to prepare the adjuvantformulations.

The PHAD formulations can be prepared at any concentration but aretypically prepared at about 0.005 to about 1.0 mg/ml of PHAD, preferably0.05 to about 1.0 mg/ml of PHAD, but preferably not more than about 2.5mg/ml of PHAD. The phosphatidylcholine of the PHAD formulation can beprepared at any concentration, but is preferably prepared at about 0.005to about 16 mg/mL (0.007 mM to 22 mM), more preferably about 0.05 toabout 8 mg/ml (0.07 mM to 11 mM), and even more preferably about 0.05 toabout 0.8 mg/ml (0.07 mM to 1 mM).

The PHAD formulations of the invention are prepared as follows toproduce a molar ratio of about 40:1 to about 1:1 of phosphatidylcholineto PHAD, preferably DPPC to PHAD, preferably about 2.5:1 DPPC to PHAD.PHAD is weighed out into an appropriate glass vial, such as a Type 1Plus Schott glass vial. An appropriate amount of phosphatidylcholine,preferably DPPC, in ethanol is added. This preparation is sonicated forapproximately 1 minute while gently swirling the preparation, and thenthe ethanol is appropriately removed via evaporation. The film isreconstituted with citrate, succinate or phosphate buffer, preferably 10mM to about 50 mM trisodium citrate, pH 6.0, and sonicated atapproximately 50° C. to 65° C., preferably 55° C., for approximately 30minutes, and more than one cycle of sonication can be performed, aspreferred. The prepared PHAD formulation typically has particles sizesfrom about 60 nm to about 500 nm. To enable sterile filtration, the PHADformulation is preferably further processed to achieve a reduced andappropriate homogenous particle size, preferably between about 70 nm to130 nm. The PHAD formulations are extruded through an 80 nm porepolycarbonate membrane (Avestin, LFLM-80) with an Avestin extruder, orequivalent, for about 7 to about 12 passes at about 45° C. to 65° C.,preferably 55° C. to achieve a homogenous particle size below about 130nm. To ensure an acceptable recovery after sterile filtration,preferably the particle sizes of the PHAD formulations are 150 nm orless, but even more preferably 130 nm or less.

The PHAD formulations may optionally then be diluted with citrate,succinate or phosphate buffer at concentrations described herein, butpreferably at about 25 mM to about 50 mM, more preferably 28 mM to about50 mM, and most preferably 30 mM to about 50 mM, pH 6.0, containing anappropriate amount of polysorbate 80 to achieve a final concentration ofpreferably 0.02% of polysorbate 80, but 0.01% to 0.1% polysorbate 80 isacceptable. Typically, the PHAD formulations will be diluted to about aconcentration of 0.05 mg/ml to 0.5 mg/mL. The PHAD formulations are thensterile filtered through a 0.2 um filter, preferably Sartorius. The PHADand adjuvant formulations of the invention described herein exhibit zetapotentials of about −20 mV to −80 mV.

The PHAD formulations described herein, in one embodiment are used inthe preparation of vaccines and are administered to animals and humansas an adjuvant of vaccines. Preferably, the formulations are designed todeliver about 10 micrograms of PHAD per dose to about 50 micrograms ofPHAD per dose, preferably about 20 micrograms of PHAD per dose to about50 micrograms of PHAD per dose, and even more preferably about 40micrograms of PHAD per dose to about 50 micrograms of PHAD per dose.

In a preferred embodiment, PHAD is provided as a single compound ofapproximately 98% purity with a molecular weight of 1763 Daltons (thestructure of which is shown in FIG. 1). One source for the preferredPHAD is Avanti Polar Lipids (Alabaster, Ala., USA). The PHADcompositions of the present invention, however, encompass phosphorylatedhexaacyl disaccharide or pharmaceutically acceptable salts ofphosphorylated hexaacyl disaccharide. The PHAD used in the compositionsmay be fully or partially synthetic or non-synthetic, although fullysynthetic is preferred.

In other embodiments, derivatives of phosphorylated hexaacyldisaccharide, such as Monophosphoryl 3-Deacyl Lipid A (available fromAvanti Polar Lipids; referred to as 3D-PHAD) are used alone or incombination with PHAD in the compositions and formulations of thepresent invention.

PHAD's purity is in stark contrast to GSK's monophosphoryl lipid Aisolated from Salmonella minnesota that exists as a dynamic, complexmixture of hexa-, penta-, and tetraacyl analogues; each of theseanalogues differ in biological activity. Those skilled in the art ofdrug and vaccine development know that PHAD is superior to GSK'smonophosphoryl lipid A because PHAD's manufacturing process, supply,use, and stability can be closely monitored and controlled as a purecompound.

The concentration of citrate, succinate, or phosphate buffer is used toimprove stability of the PHAD formulation at room temperature and up toabout 37° C. While not bound by theory, it is believed that asynergistic effect results from composition of molar ratio of PC:PHAD,which is a preferred combination, and citrate, succinate, or phosphatebuffers within a specific concentration range as described herein.

Importantly, the PHAD formulations are preferably formulated to besubstantially free of various excipients or chemicals. Therefore, theaddition of cholesterol or two or more phosphatidylcholines or one ormore phosphatidylglycerols is not required, and preferably not includedin the formulations of the invention. Adjuvant formulations essentiallyfree of metabolizable oils, including squalene, and substantially freeof cholesterol are preferred. In addition, while the prior art suggestscholesterol is a necessary chemical required for liposomes or adjuvantformulations, the adjuvant formulations described herein do not requirecholesterol for the adjuvant to provide all the significant advantagesover prior art formulations, and preferably no cholesterol is added tothe adjuvant formulations. As shown in the Examples, two or morephosphatidylcholines or one or more phosphatidylglycerols (for a totalof two or more phosphatidylcholines or phosphatidylglycerols) mayoptionally be added to the formulations at minor concentrations, butthese are unnecessary and not required to achieve stability at roomtemperature or up to about 37° C. of the invention or to prevent, lower,or reduce severe injection site and systemic reactions while enhancingan immune response.

As detailed herein, those skilled in the art have been working withliposomes and other various formulations containing MLA, MPL, orsynthetic analogues of these adjuvants for more than twenty years andhave been unable to produce a formulation that allows for stability asdescribed herein while in aqueous suspensions. Room temperature and upto about 37° C. stability for vaccine adjuvants is a highly sought aftergoal by those skilled in the art. Despite the efforts, no formulationsto keep MLA, MPL, or synthetic analogues stable at the temperatures andfor the time periods described herein have been developed. The inventiondescribed herein solves this problem.

Preparation of Vaccines

Another embodiment of this invention further describes novel vaccinecompositions comprising the adjuvant or PHAD formulations and FimCH ortruncated FimH. Such vaccines are used to treat and prevent urinarytract infections caused by gram-negative bacteria including Escherichiacoli and multi-drug resistant E. coli. FimCH is a non-covalent complexof FimC and FimH recombinant proteins. A vaccine of FimCH and PHADformulation is prepared by adding a predetermined volume of a PHADformulation to a vial of FimCH.

The following is an example of a vaccine prepared in accordance to theinvention. Generally, to prepare a vaccine for administration, aneffective amount of an antigen of FimCH or truncated FimH is combinedwith an adjuvant formulation containing about 0.005 mg/ml to about 0.5mg/ml of PHAD to administer about 10 μg to about 50 μg of PHAD perinjection to a human.

In practice, the following is an example of one procedure that may beused by medical personnel to prepare and administer a vaccine inaccordance with the invention. The conditions and procedures areexemplary and do not limit the scope of the invention.

A vial of FimCH vial is removed from storage of about −20° C. andallowed to stand at room temperature for approximately twenty minutes toreach approximate room temperature. After the FimCH vial reachesapproximately room temperature, the vial is inverted a number of timesto mix the contents. Separately, a vial containing the PHAD formulationis removed from a storage container from 2° C. to 8° C. storage. Thevial of PHAD formulation is inverted a number of times to mix thecontents, and then about 0.2 mL is withdrawn with a sterile 1.0 mLsyringe and injected into the FimCH vial through the stopper. Again thevial is inverted a number of times to mix the contents. Sterile waterfor injection (WFI) or more preferably preferred sterile buffer of theinvention is withdrawn in an amount of 0.2 mL using a sterile 1.0 mLsyringe and injected into the FimCH/PHAD vial through the stopper. Againthe vial is inverted a number of times. Finally, about 0.3 mL ofprepared FimCH/PHAD vaccine using a sterile 1.0 mL syringe is withdrawn.The prepared vaccine contains 50 μg of FimCH and 20 μg of PHAD per a 0.3mL dose. This prepared vaccine can be stored at refrigerated or roomtemperatures prior to administration.

In general FimCH is stored at −70° C., −20° C., or 2° C. to 8° C. forlong time periods, or even room temperature for a short time period ofabout 4 days to two to three weeks. Typically, only sterile productsshould be stored at room temperature because if the products are notsterile, microbial growth is possible, but not guaranteed. The vaccineis preferably administered by intramuscular injection. Typically, about5 micrograms of FimCH to about 200 micrograms of FimCH would beadministered to a human, preferably about 20 micrograms to about 110micrograms. Typically, about 10 micrograms of PHAD per dose to about 50micrograms of PHAD per dose would be administered with FimCH, morepreferably about 20 micrograms of PHAD per dose to about 50 microgramsof PHAD per dose, even more preferably about 40 micrograms of PHAD perdose to about 50 micrograms of PHAD per dose although more or less maybe used. Typically, three to four doses of FimCH with the PHADformulation is administered to a patient in need. These doses typicallyoccur at day 0 and then about days 30 to 60, then about 90 to 180 days,and then, if preferred, about 180 to 360 days from the firstadministration. As needed, additional injections may occur 12 to 36months after the initial vaccination.

As described above, the more preferable aspect of the invention is thatthe PHAD compositions and formulations are stored separately from theantigen or FimCH because the PHAD compositions and formulations haveexcellent stability, and formulated or mixed appropriately with theantigen or FimCH sometime before administration to a patient or human.However, other methods of mixing, preparing and administering vaccinesare possible and will function effectively.

The data in the following sections demonstrates that the adjuvantformulations of the invention enhance the immune response of otherantigens including bacterial and viral antigens. One or more vaccineantigens may be added to the PHAD formulations prepared in accordancewith the invention. These antigens may be the FimCH protein complex asdescribed herein or other antigens including, but not limited todiphtheria, tetanus, pertussis, poliomyelitis, hepatitis, and orantigenic preparations of the influenza virus.

In another embodiment, methods of administration of the novel vaccinecompositions comprising adjuvant or PHAD formulations and FimCH ortruncated FimH are provided. Particularly, methods of treatment toprevent and treat urinary tract infections caused by gram-negativebacteria including E. coli and multi-drug resistant E. coli areprovided. Typically, about 5 micrograms of FimCH to about 200 microgramsof FimCH would be administered to a human, preferably about 20micrograms to about 110 micrograms. Typically, about 10 micrograms ofPHAD per dose to about 50 micrograms of PHAD per dose would beadministered, preferably about 20 micrograms of PHAD per dose to about50 micrograms of PHAD per dose, even more preferably about 40 microgramsof PHAD per dose to about 50 micrograms of PHAD per dose. Other dosageamounts and regimens may be used dependent on the antigen used and thecondition being treated.

In another embodiment, methods of inducing the production of antibodiesagainst FimH in a human with recurrent urinary tract infections areprovided.

In another embodiment, vaccine compositions that induce the productionof antibodies against FimH in a human with recurrent urinary tractinfections are provided.

In another embodiment, sterile compositions and sterile pharmaceuticalcompositions containing PHAD are provided; preferably these compositionsof aqueous buffered suspensions have particle sizes less than 150 nm,even more preferably less than 130 nm. The sterile PHAD compositions andformulations are stored in a pharmaceutical container which is in directcontact to the PHAD compositions and formulations. Examples of thesepharmaceutical containers are vials or syringes, more preferably thesterile PHAD compositions and formulations are contained in a sterilesyringe. These pharmaceutical containers holding the PHAD composition orformulation can be stored in a temperature-validated container, e.g.incubator, at room temperature. These pharmaceutical containers holdingthe sterile PHAD composition formulation can be added to a shippingcontainer assembled to be transferred to another location at roomtemperature or up to about 37° C. These shipping containers can betransferred via a government postal service or a commercial shippingservice. Due to the remarkable stability of the inventions describedherein, the location receiving said PHAD compositions and formulationsmay be a location without refrigeration or intermittent access torefrigeration or a location without electricity or with intermittentelectricity.

In another embodiment, a vaccine kit comprising the PHAD or adjuvantcompositions or formulations or vaccine composition is provided. The kitmay optionally include methods of preparing and administering thevaccine and/or instructions for storage and exposure of the PHADcompositions or formulations at room temperature and up to and about 37°C. These instructions describing storage, shipping, and exposuretemperatures may be approved by a government regulatory authorityincluding the US FDA or European Medicines Agency. Preferably, one ormore of the kit components is the PHAD compositions or formulations in asyringe. Preferably the PHAD compositions or PHAD containingformulations are sterile and are in a sterile syringe.

The kit may include a label for the PHAD or adjuvant compositions orformulations of the invention providing instructions or limitations forstorage and exposure of the PHAD compositions or formulations at roomtemperature and up to and about 37° C. The labels or instructionsdescribing storage, shipping, and exposure temperatures may be approvedby a government regulatory authority including the US FDA or EuropeanMedicines Agency.

As stated herein, the novel characteristics of the invention enable thePHAD compositions and formulations to be manufactured, tested, analyzed,stored, shipped, held, moved, transferred, or administered atrefrigerated temperatures, room temperature, up to and about 37° C.,temperatures between refrigerated temperatures and room temperature forperiods of time as described herein. Due to the remarkable stability ofthe inventions described herein, the location receiving said PHADcompositions and formulations may be a location without refrigeration orintermittent access to refrigeration or a location without electricityor with intermittent electricity.

EXAMPLES

Certain specific aspects and embodiments of the present disclosure willbe explained in more detail with reference to the following examples,which are provided solely for purposes of illustration and are not to beconstrued as limiting the scope of the disclosure in any manner. Theamounts used do not represent a limitation and the process can be scaledup to produce larger batches

Example 1

PHAD formulations are manufactured as follows to produce a molar ratioof about 2.5:1 of DPPC to PHAD.

PHAD, phosphorylated hexaacyl disaccharide, and DPPC can be obtainedfrom Avanti Polar Lipids (Alabaster, Ala., USA) as either non-GMP or GMPmaterial (the Certificate of Analysis is provided in Table 1). PHAD is asynthetic version of monophosphoryl lipid A. PHAD is formulated withDPPC to prepare the adjuvant formulation of the invention. DPPC'srelease specifications are provided in the table 2. The transitiontemperature of DPPC is 41° C.

PHAD is weighed out into an appropriate glass vial, preferably a Type 1Plus Schott glass vial. An appropriate amount of an approximately 2.3mg/ml DPPC solution, or equivalent, in ethanol is added. Thispreparation is sonicated for approximately 1 minute while gentlyswirling the preparation, and then the ethanol is appropriately removedvia evaporation using care. The film is reconstituted with 10 mMtrisodium citrate, pH 6.0, and sonicated at approximately 50° C. to 65°C., preferably 55° C., for approximately 30 minutes. The PHADformulations are typically prepared at about 0.5 to 1.0 mg/ml, but notmore than about 2.5 mg/ml. (As described herein, molar ratios of forexample about 13:1 DPPC:PHAD can also be prepared using this sameprocedure by adjusting the amount of DPPC as needed.

The prepared PHAD formulations typically exhibit particles sizes fromabout 60 nm to about 500 nm. To enable sterile filtration, the PHADformulations have to be further processed to achieve a reduced andappropriate homogenous particle size, typically between about 70 nm to130 nm. Even though numerous literature references, including U.S. Pat.No. 6,630,161, report high-pressure homogenization is a preferred methodto reduce the particle size of liposomes, high-pressure homogenizationusing an Avestin homogenizer of pressures up to 25,000 psi do notsignificantly or relevantly reduce the particle sizes of these PHADformulations. This difficulty was unexpected. These PHAD formulationsmust be extruded through an 80 nm pore polycarbonate membrane (Avestin,LFLM-80) with an Avestin extruder, or equivalent, for about 7 to about12 passes at about 45° C. to 65° C., preferably 55° C. to achieve ahomogenous particle size below about 130 nm. Extruding at 45° C. to 65°C. is an important parameter. To ensure an acceptable recovery aftersterile filtration, the particle sizes of the PHAD adjuvant formulationsare preferably 150 nm or less, but more preferably 130 nm or less.

The PHAD formulations may then be diluted with 10 mM trisodium citrate,pH 6.0 containing an appropriate amount of polysorbate 80 to achieve afinal concentration of most preferably 0.02% of polysorbate 80, but0.01% to 0.1% polysorbate 80 is acceptable. Typically PHAD formulationswill be diluted to about a concentration of 0.05 mg/ml to 0.5 mg/mL.These PHAD formulations are then sterile filtered through a 0.2 umfilter, preferably Sartorius.

As determine by cryogenic transmission electron microscopy, the adjuvantformulations described herein are suspensions.

Additional or alternative steps may be added to the procedure above toprepare the PHAD formulations. For one example, the ethanol may beevaporated by rotary evaporation, or equivalent, or via a nitrogenstream, or equivalent. For another example, the sonication stepincluding a buffer of the invention may be repeated two or more times,the formulation may be cooled to room temperature or less betweenrepeated sonications, and the formulation may be held at a temperaturesimilar to the sonication step for one or more hours before, during, orafter said sonication.

TABLE 1 Certificate of Analysis Information for PHAD from Avanti PolarLipids, Inc. Analysis Specification Results Physical examination Whiteto off-white powder Pass or lyophilized cake which contains no foreignmatter. TLC Ninhydrin spray, negative All pass (65:25:4 (v/v/v) Iodine,1 major spot chloroform:methanol:water) Phosphorus spray, positiveCharring, positive Water dip, 1 major spot Rf consistent with structureHPLC NLT 97% purity 99.2% Proton NMR Consistent with structureConsistent with structure Phosphorus NMR Consistent with structureConsistent with structure MS Consistent with structure Consistent with(exact mass = 1762.3) structure Karl Fischer Water NMT 5% water  1.4%Titration Residual Solvents NMT 2000 ppm methanol None Detected(GC/FID)* NMT 2000 ppm ethanol None Detected NMT 2000 ppm acetone NoneDetected NMT 200 ppm hexane None Detected NMT 2000 ppm None Detectedcyclohexane NMT 500 ppm toluene None Detected NMT 50 ppm chloroform/None Detected ethyl acetate NMT 5000 ppm total None Detected residualsolvents Palladium (ICPMS)* NMT 10 ppm <0.1 ppm Heavy Metals Screen +NMT 20 ppm  <20 ppm Ruthenium + Iridium by ICPMS*

TABLE 2 Certificate of Analysis Information for DPPC from Avanti PolarLipids, Inc. Analysis Specification Results Physical examination Whitesolid which contains Pass no foreign matter. TLC Ninhydrin spray,negative All Pass Iodine, 1 major spot Phosphorus spray, positiveCharring, negative Water dip, 1 major spot Rf consistent with structureQuantitative Phosphorus NMT 1% 16:0 dimethyl PE None detected NMR HPLCNMT 1% palmitic acid None detected NMT 1% 16:0 lyso PC 0.7% NLT 99%purity 99.3% Fatty acid methyl ester NLT 99% (AUC) palmitoyl 100.0% byGC/FID methyl ester Karl Fischer Water NMT 8% water 0.5% TitrationResidual Solvents by NMT 100 ppm methanol None Detected GC/FID NMT 100ppm ethanol None Detected NMT 100 ppm acetone None Detected NMT 100 ppmhexane None Detected NMT 100 ppm cyclohexane None Detected NMT 100 ppmtoluene None Detected NMT 20 ppm chloroform None Detected NMT 250 ppmtotal residual None Detected solvents

Example 2

Antigens for the vaccine may be prepared as follows.

FimCH is a non-covalent complex of FimC and FimH recombinant proteins.The recombinant proteins are derived from transgenic E. coli culture.The FimC and FimH proteins are expressed separately in E. coli, and theyspontaneously form a non-covalent complex. The molecular weight of theFimCH complex is approximately 51,700 Daltons.

The FimH protein (SEQ ID No: 1) of the complex has a molecular weight of29,065 Daltons, and it consists of 279 amino acid residues representedby the sequence below:

Phe Ala Cys Lys Thr Ala Asn Gly Thr Ala Ile ProIle Gly Gly Gly Ser Ala Asn Val Tyr Val Asn LeuAla Pro Val Val Asn Val Gly Gln Asn Leu Val ValAsp Leu Ser Thr Gln Ile Phe Cys His Asn Asp TyrPro Glu Thr Ile Thr Asp Tyr Val Thr Leu Gln ArgGly Ser Ala Tyr Gly Gly Val Leu Ser Asn Phe SerGly Thr Val Lys Tyr Ser Gly Ser Ser Tyr Pro PhePro Thr Thr Ser Glu Thr Pro Arg Val Val Tyr AsnSer Arg Thr Asp Lys Pro Trp Pro Val Ala Leu TyrLeu Thr Pro Val Ser Ser Ala Gly Gly Val Ala IleLys Ala Gly Ser Leu Ile Ala Val Leu Ile Leu ArgGln Thr Asn Asn Tyr Asn Ser Asp Asp Phe Gln PheVal Trp Asn Ile Tyr Ala Asn Asn Asp Val Val ValPro Thr Gly Gly Cys Asp Val Ser Ala Arg Asp ValThr Val Thr Leu Pro Asp Tyr Arg Gly Ser Val ProIle Pro Leu Thr Val Tyr Cys Ala Lys Ser Gln AsnLeu Gly Tyr Tyr Leu Ser Gly Thr His Ala Asp AlaGly Asn Ser Ile Phe Thr Asn Thr Ala Ser Phe SerPro Ala Gln Gly Val Gly Val Gln Leu Thr Arg AsnGly Thr Ile Ile Pro Ala Asn Asn Thr Val Ser LeuGly Ala Val Gly Thr Ser Ala Val Ser Leu Gly LeuThr Ala Asn Tyr Ala Arg Thr Gly Gly Gln Val ThrAla Gly Asn Val Gln Ser Ile Ile Gly Val Thr Phe Val Tyr Gln

The FimC (SEQ ID No: 2) protein of the complex has a molecular weight of22,700 Daltons, and it consists of 205 amino acid residues representedby the following sequence:

Gly Val Ala Leu Gly Ala Thr Arg Val Ile Tyr ProAla Gly Gln Lys Gln Val Gln Leu Ala Val Thr AsnAsn Asp Glu Asn Ser Thr Tyr Leu Ile Gln Ser TrpVal Glu Asn Ala Asp Gly Val Lys Asp Gly Arg PheIle Val Thr Pro Pro Leu Phe Ala Met Lys Gly LysLys Glu Asn Thr Leu Arg Ile Leu Asp Ala Thr AsnAsn Gln Leu Pro Gln Asp Arg Glu Ser Leu Phe TrpMet Asn Val Lys Ala Ile Pro Ser Met Asp Lys SerLys Leu Thr Glu Asn Thr Leu Gln Leu Ala Ile IleSer Arg Ile Lys Leu Tyr Tyr Arg Pro Ala Lys LeuAla Leu Pro Pro Asp Gln Ala Ala Glu Lys Leu ArgPhe Arg Arg Ser Ala Asn Ser Leu Thr Leu Ile AsnPro Thr Pro Tyr Tyr Leu Thr Val Thr Glu Leu AsnAla Gly Thr Arg Val Leu Glu Asn Ala Leu Val ProPro Met Gly Glu Ser Ala Val Lys Leu Pro Ser AspAla Gly Ser Asn Ile Thr Tyr Arg Thr Ile Asn AspTyr Gly Ala Leu Thr Pro Lys Met Thr Gly Val Met Glu

For producing the transgenic cell line, the FimC gene from E. colistrain J96 was amplified with primers SLC4-28-fimC5 and SLC4-28-FimC3from purified J96 genomic DNA to give a 771 base pair product. This wasdigested with BamHI and EcoRI, purified, and ligated into pTRC99a cutwith the same enzymes (BamHI and EcoRI). The ligation product wastransformed into E. coli C600 cells, and selected on ampicillin,producing plasmid pSJH-32.

The ampicillin antibiotic resistance was switched to kanamycin using thefollowing procedure: Primers pKD4-pr1 and pKD4-pr2 were used to amplifythe kanamycin resistance gene from pKD4. This PCR product wasphosphorylated with T4 polynucleotide kinase and gel purified. pSJH-32was cut with ScaI and BglI, blunted with T4 DNA polymerase,dephosphorylated with calf intestinal alkaline phosphatase, then ligatedwith the phosphorylated kanamycin resistance gene PCR product. Theligation product was then transformed into E. coli C600 cells andselected on kanamycin, creating plasmid pSJH-319.

The FimH gene from strain J96 was amplified with primers FimH5 and FimH3from purified J96 genomic DNA to give a 978 base pair product. It wasdigested with SacI and HindIII, purified, and ligated into pBAD33digested with SacI and HindIII. Afterwards the construct was transformedinto C600 cells, and selected on chloramphenicol.

Example 3

Bioprocessing (Process to obtain antigens of the vaccine).

The bioprocessing step is initiated with inoculation of master cell bank(MCB) into shake flasks containing APS LB medium with kanamycin (50μg/mL) and chloramphenicol (20 μg/mL). When the OD reaches 2.0-3.0 units(after approximately 15 hours growth), the cell culture is transferredaseptically into reactors for fed-batch fermentation. The mediumcontaining APS Super Broth, about 0.8% glycerol, and antibiotics issterilized prior to inoculation. FimH protein expression is induced atOD≧10 with IPTG. Five minutes after IPTG addition, FimC proteinexpression is induced with arabinose. Cells are harvested approximatelyone hour later. After harvesting, the cells are separated from the mediacomponents by continuous or batch centrifugation.

Protein Recovery

Recombinant FimCH is expressed in the E. coli periplasm. E. coli, as agram negative bacteria, possess an inner and an outer lipid bilayermembrane. The space between the lipid bilayers is the periplasm.Immediately after centrifugation, FimCH is recovered from the cell usinga periplasm preparation. The cells are reacted with recombinant lysozymein the presence of sucrose, Tris, and EDTA at 2-8° C. The mixture isthen centrifuged, and the resulting periplasmic protein solution iscollected. The protein is then precipitated with ammonium sulfate,centrifuged, resuspended in 20 mM MES pH 5.9, and diafiltered viadialysis into 20 mM MES pH 5.9 using SpectraPor 2 Dialysis Membrane(Spectrum Labs 132680). When the solution conductivity decreases toapproximately ≦1.5 mS/cm, the solution is collected and transferred topurification.

Protein Purification

Purification consists of three column chromatography steps (1.CEX, 2.HIC, 3. CEX), one buffer exchange step via diafiltration usingSpectraPor 2 Dialysis Membrane (Spectrum Labs 132680) followed byfiltration, and one final aseptic filtration step. The diafiltrationstep is used to exchange the protein into 20 mM MES buffer pH 5.9 sothat it will bind the second CEX column.

The two CEX steps use Source 15S (GE Healthcare 17-1273-02) in an XK26column. For both CEX columns the following conditions are used: BufferA: 20 mM MES, pH 5.9; Buffer B: 20 mM MES/500 mM Sodium Chloride, pH5.9; 8 ml/min for all steps except loading for XK26/10 column,pre-equilibrate the column with 5 CV of Buffer B, equilibrate the columnwith 4 CV of Buffer A, load Dialyzed FimCH sample at 5 ml/min forXK26/10 using a sample pump and specifically not via the chromatographypump, wash the column with 4 CV of Buffer A, and elute the column with alinear 5 CV gradient from 0-25% Buffer B with fraction collection.

For the HIC column used Butyl Sepharose 4FF (GE Healthcare 17-0980-01)in XK26 column. Buffer C: 20 mM MES/550 mM Ammonium Sulfate, pH 5.9; 8ml/min for XK26/10 except loading, pre-equilibrate the column with 3 CVof Buffer A (as above), equilibrate the column with 6 CV of Buffer C,load Pooled FimCH sample at 5 ml/min XK26/10 column, wash the columnwith 6 CV of Buffer C, elute the column with a linear 4 CV gradient from0-100% Buffer A with fraction collection. FimCH is formulated in theconcentration of 0.3 mg/mL in 20 mM MES pH 5.9 or 20 mM trisodiumcitrate, pH 5.4. It is then aseptically filtered through a 0.2 μmsterile filter. FimCH is stable and can be stored at −20° C. for aboutat least 2 years.

Example 4

Potency by In Vitro Mannose Binding (Demonstrates the biologicalactivity of FimCH).

The biological activity of the FimCH drug substance (for example, fromExample 3) is determined by an in vitro mannose binding assay. The FimHprotein is a bacterial adhesin utilized by E. coli to bind mannoseresidues on glycosylated proteins. During urinary tract infections, theFimH adhesin binds mannosylated uroplakin proteins on bladder epithelialcells, which promotes the internalization of bound E. coli. The bindingof FimH to mannosylated uroplakin is essential for E. coli to causeurinary tract infections. To monitor the mannose-binding activity ofFimH in vitro, FimH binding to the enzyme horseradish peroxidase (HRP)is observed. HRP is a glycosylated protein containing mannose residues,and has previously been used to study mammalian mannose-bindingreceptors. Complexes of HRP and the lectin ConA, which bindsα-D-mannosyl and α-D-glucosyl groups, have also been generated andstudied. These results demonstrate that HRP acts as a ligand for otherknown mannose-binding proteins. Using this potency assay as describedbelow, HRP binding to FimH is shown to be concentration-dependent and tobe inhibited by small molecules that the block the binding of mannose toFimH.

In the in vitro FimCH potency assay, FimCH is “captured” by purified andqualified anti-FimH antisera bound to an ELISA plate. The anti-FimHantisera used in this assay have demonstrated the ability to bind toFimH in both indirect ELISAs (as the detection antisera, FIG. 3) andwestern blots. HRP is then added, excess HRP is rinsed away, and theactivity of bound HRP is detected. The measured HRP activity isproportional to the concentration of FimCH added (FIG. 4). These resultsdemonstrate that HRP binds to FimH in a dose-dependent manner.

To demonstrate that HRP binding to FimH requires FimH mannose-bindingactivity, a mannose binding-deficient FimH mutant named Q133K, which isalso in complex with FimC, was analyzed and compared to FimCH. Q133Kshares the same amino acid sequence as FimH, except that a criticalglutamine at position 133 is replaced by lysine. This mutation is in theFimH mannose-binding pocket and renders Q133K functionally deficient inbinding mannose and mannosylated proteins. As shown in FIG. 4, Q133KFimCH does not bind HRP. In an indirect ELISA (FIG. 3), the Q133K mutantcomplex is recognized by the purified anti-FimH antisera. Thisdemonstrates that the lack of HRP signal with Q133K is not due to aninability of the purified antisera to bind to Q133K; instead, it is dueto the inability of Q133K to bind mannose residues on HRP. These resultsalso demonstrate that HRP does not bind FimC, because Q133K is also incomplex with FimC.

As demonstrated in Hung et al. 2002, single point mutations in FimH atpositions 54, 133, 135, and 140 completely abolish mannose binding. Asreported by Hung et al., “ . . . even the slightest change in themannose-binding pocket, in an atom that does not bind directly tomannose, significantly reduces binding,” suggesting that mutations thatcould occur in vitro could severely limit or abolish FimH mannosebinding activity. The lack of HRP binding to the Q133K mutant supportsthis assay's ability to assess the biological activity of FimH bindingto mannosylated proteins.

Several small molecule inhibitors of mannose binding to FimH have beendescribed. Two of these inhibitors,4-methylumbelliferyl-α-D-mannopyranoside (UFMP) andmethyl-α-D-mannopyranoside (MDMP), have been used to further qualifythis potency assay. The reported dissociation constant (Kd) for UFMPbinding to FimH is 20 nM, which is approximately 100-fold more potentthan MDMP's Kd of 2.2 μM. As expected, the addition of either of theseFimH mannose binding inhibitors in the HRP binding step blocks HRPbinding to FimH in a dose-dependent manner (see FIG. 5). For UFMP, 50%inhibition is observed at 10 ng/mL (30 nM). For MDMP, 50% inhibition isobserved at approximately 1 μg/mL (5.1 μM), which is approximately100-fold higher than the concentration of UFMP.

These results demonstrate this assay's ability to assess the biologicalactivity of FimH and verify the consistency of the manufacturing processfrom batch to batch. Furthermore, this potency assay confirms the properfolding of the FimH epitope and is predictive of the generation of IgGanti-FimH that has been shown to reduce E. coli CFU in bladders of miceby the administration of the FimCH/PHAD vaccine.

Example 5 Impurities of FimCH Drug Substance by CEX-HPLC

CEX-HPLC is used for determination of the FimCH complex, unbound FimCand impurities in the final FimCH drug substance. The protein is elutedfrom a GE Healthcare Mono S 5/50 GL column using a gradient of 0.3 MNaCl in 20 mM MES buffer, pH 6.2 (Buffer B) (Buffer A is 20 mM MESbuffer, pH 6.2). At T=0 the mobile phase is 100% Buffer A, and at T=22minutes, the mobile phase is 100% Buffer B. The relative content of theunbound FimC and impurities is determined based on the peak areas. Arepresentative chromatogram is provided in FIG. 6.

Example 6

FimCH and PHAD formulation: An 85-Day IntramuscularToxicity/Immunogenicity Study in Rabbits with a 21 Day Recovery Period(GLP).

The pivotal GLP toxicity study to evaluate the toxicity andimmunogenicity of the FimCH vaccine containing a PHAD formulation of theinvention (DPPC:PHAD—about 2.4:1 molar ratio) was conducted in femalerabbits. The study examined ophthalmological findings, antibodyassessment, and histopathology. Female rabbits were administered a totalof 5 doses of saline control (N=6), PHAD alone at 40 to 50 μg (N=12),FimCH at 100 μg plus 20 μg PHAD (N=6; low dose), or FimCH at 125 μg plus40 to 50 μg PHAD (N=12; high dose) via IM injection every 3 weeks (Days1, 22, 43, 64, and 85) for 13 weeks. Three days following the fifth dose(Day 88), 6 rabbits per group were euthanized with the remaining 6rabbits in the PHAD alone group and FimCH high dose groups euthanizedfollowing a 3-week recovery period (Day 106).

Toxicity was assessed based on clinical observation, ophthalmology, bodytemperature, body weight, food consumption, clinical pathology, grossnecropsy, organ weight, and histopathology data. Body temperatures wereobtained prior to and 2, 4, 6, 24, 48 and 72 hours after each injection.In addition to standard clinical pathology parameters (predose, Days 2and 88), C-reactive protein (CRP) and fibrinogen were evaluated 2 and 7days after dosing. Potential injection site reactions were scored foredema and erythema using the Draize scale and assessed for othermanifestations of local toxicity (i.e. eschar, vesiculation, ulceration,and hematoma) 24, 48, and 72 hours post dose. Anti-FimH antibodyassessment was performed on serum samples collected prior dosing on Days1, 22, 43, 64 and 85, and prior to necropsy on Days 88 and 106, on urinesamples collected prior to dosing, Day 64, and prior to necropsy on Days88 and 106, and on vaginal washings collected prior dosing on Days 1and, and prior to necropsy on Days 88 and 106. Antibody evaluation ofthe urine and vaginal wash samples was qualitative using a qualifiedMSD-ECL assay. Antibody levels in serum were determined using avalidated MSD-ECL assay. Of 18 rabbits vaccinated with FimCH and PHAD,17 demonstrated anti-FimH IgG titers at about 1:3,200,000 at about Day88.

All rabbits survived to scheduled necropsy. Preliminary data indicatedthat FimCH plus PHAD as well as PHAD alone were well tolerated. Thefindings demonstrated that there were no apparent PHAD alone orvaccine-related effects on clinical observations, body weight, foodconsumption, body temperature, clinical pathology, or organ weights.Local reaction, based on in-life observations, was limited.

A similar rabbit study was performed using FimCH with PHAD prepared asDPPC:PHAD at about a molar ratio of 1:3.9. In this study, only 5 of 16rabbits at about Day 43 demonstrated anti-FimH IgG titers from 1:400,000to 1:800,000. In contrast, 16 of 18 rabbits vaccinated with FimCH withPHAD prepared as DPPC:PHAD at about a molar ratio of 2.4:1 (listedabove) demonstrated anti-FimH IgG titers from 1:400,000 to 3,200,000 atabout Day 43. These immunogenic differences are consistent with thestudies performed in mice described herein demonstrating that a molarratio of DPPC:PHAD of about 2.4:1 is superior to a molar ratio ofDPPC:PHAD of about 1:3.9.

Example 7

To test the efficacy of PHAD as an adjuvant for systemic vaccination inthe mouse UTI infection model, C3H/HeN mice were infected withapproximately 1×10⁸ CFU of clinical cystitis E. coli isolate UTI89 viatransurethral catheterization after IM immunization. Female C3H/HeN mice(approximately 9 weeks old) were purchased from Charles Riverlaboratories (Wilmington, Mass.). Mice were injected via theintramuscular route (IM) in the right thigh under light isofluraneanesthesia (Henry Schein, Melville, N.Y.) in a 50 μl volume using a 30gauge needle. In these efficacy studies, mice were immunized with 12.5μg PHAD/15 μg FimCH. Mice immunized using PHAD as the adjuvant and FimCHas antigen showed a statistically significant decrease of E. coli CFU inbladders one or two days after infection compared to mice immunized withadjuvant alone and to naïve mice (experiment using PHAD shown in FIG.7). These data demonstrate that a FimCH vaccine adjuvanted with PHADproduces antibodies in mice that reduce the E. coli colonization ofbladders. These data offer evidence that a FimCH vaccine adjuvanted withPHAD prepared according to the compositions described herein and usedaccording to the methods described herein administered to a humanpatient in need will also reduce the E. coli colonization of bladders inhumans.

Example 8

Truncated FimH (FimHt) adjuvanted with PHAD formulation or Freund'sadjuvant: An Immunogenicity Study in Rabbits.

Female rabbits were administered a total of 3 doses of FimHt at 100 μgplus about 50 μg PHAD formulation of the invention (N=2) on days 0, 21,and 42, or a total of 5 doses of FimHt at 100 μg plus Freund's adjuvant(complete for initial vaccination and incomplete for each boost at aboutdays 14, 21, 49, and 70) (N=2) via IM injection. In this experiment,truncated FimH has a series of histidines or histidine tag, and thoseskilled in the art understand that other truncated versions of FimH canalso be used, most preferably requiring the mannose binding domain.Truncated FimH in this example consists of FimH residues 1 to 175 with aC-terminal 6-histidine tag (SEQ ID No: 3). The sequence of FimH isdescribed in Example 2. Anti-FimH antibody assessments were performed onserum samples collected on about day 30 or about days 35 and 56.Antibody levels in serum were determined using an ELISA as describedherein. The capture antigen for this experiment was an equivalenttruncated FimH without a histidine tag. Rabbits vaccinated with bothformulations demonstrated anti-FimH IgG greater than 1:1,600,000(preimmune sera <1:10,000). Previous truncated versions of FimH havebeen publicly disclosed and one example is as in U.S. Pat. No.6,737,063, which is specifically incorporated in its entirety.

Example 9 FimCH Vaccine with PHAD Formulation Administered to Rabbits

Two groups of rabbits (N=3) were immunized at day 0 and boosted at days21, 42, via IM injection with 50 μg FimCH and 54 μg PHAD with andwithout 0.1% polysorbate 80 (PHAD formulation of the invention, 10 mMtrisodium citrate, pH 6.0). Antibody levels in serum were determinedusing an ELISA as described herein. Serum was collected from both groupsincluding at about day 30. IgG anti-FimH titers were approximatelyequivalent to or more than 1:1,600,000 in both groups (preimmune sera<1:10,000). The data described herein demonstrate that FimCH vaccinewith PHAD formulations of the invention with or without polysorbate 80generate an equivalent immunogenic response.

Example 10 HPLC Analysis of PHAD and DPPC in Compositions

PHAD and DPPC concentrations in the PHAD formulation are analyzed byHPLC-ELSD using an Agilent Eclipse XBD C18, 1.8 um, 4.6 mm×50 mm column.The mobile phases are as follows: MP A: 20 mM ammonium acetate/1% aceticacid in water; MP B: 20 mM ammonium acetate/1% acetic acid in methanol;and MP C: 20 mM ammonium acetate/1% acetic acid in methanol/chloroform(50/50). Method 1: gradient begins at 5% MP A and 95% MP B, at 2 minutesis 100% MP B, and at 8 minutes is 100% MP C. Method 2:gradient begins at5% MP A and 95% MP B, at 2 minutes is 100% MP B, and at 15 minutes is100% MP C. Sample Diluent 1: 85:15 (75:15:10 methanol:chloroform:waterwith 20 mM ammonium acetate/1% acetic acid):(1:1 methanol:chloroformwith 20 mM ammonium acetate/1% acetic acid) Sample and standards arediluted 1:4 with sample diluent 1. Sample Diluent 2: (70:25:5)methanol:chloroform:water with 20 mM ammonium acetate/1% acetic acid. Ifusing sample diluent 2, sample and standards are diluted 1:10 withsample diluent 2. The ELSD gain is at 8 with temperature at 60 C andnitrogen flow set to approximately 3.7 bars. An example chromatogramusing method 2 and sample diluent 2 is shown in FIG. 8.

Example 11 Stability Studies of PHAD Compositions and Formulations

Using the HPLC method as described in Example 10, the stabilities ofdifferent preparations of PHAD as a suspension or PHAD formulations as asuspension including phosphatidylcholines were monitored by analyzingthese preparations for PHAD concentration and comparing it to itsinitial results. A PASS result means the PHAD concentration was withinplus/minus 20% of the initial testing results of the release samples,which is within the limits of the HPLC method using an evaporative lightscattering detector (ELSD). PHAD concentrations of these preparationswere compared when stored at 2° C. to 8° C. or 25° C. or to 37° C. toproject (estimate) the stability of these preparation over months toyears. To those skilled in the art, data from 25° C. is anintermediate/accelerated condition and data from 37° C. is anaccelerated condition used to project a shelf life at the long termstorage condition of 2° C. to 8° C. Buffers that enabled superiorstability for PHAD were first determined; then these preferred bufferswere evaluated with a PHAD formulation including a phophatidylcholine.

TABLE 3 Stability of PHAD at 25° C. for 7 days in Select BuffersCondition/ Time Point Result 1 0.5 mg/ml PHAD in WATER 25° C./ FAIL 7days 2 0.5 mg/ml PHAD in 10 mM trisodium citrate, 25° C./ PASS pH 6.0 7days 3 0.5 mg/ml PHAD in 10 mM trisodium citrate, 25° C./ PASS pH 5.0 7days 4 0.5 mg/ml PHAD in 50 mM trisodium citrate, 25° C./ PASS pH 6.0 7days 5 0.5 mg/ml PHAD in 100 mM trisodium citrate, 25° C./ FAIL pH 6.0 7days 6 0.5 mg/ml PHAD in 10 mM sodium acetate, 25° C./ FAIL pH 6.0 7days 7 0.5 mg/ml PHAD in 10 mM disodium 25° C./ PASS succinate, pH 6.0 7days 8 0.5 mg/ml PHAD in Phosphate buffered saline 25° C./ FAIL (PBS) 7days 9 0.5 mg/ml PHAD in 200 mM Na₂HPO₄ and Not precip- 100 mM citricacid, pH 6.0 applicable itate 10 0.5 mg/ml PHAD in 20 mM Na₂HPO₄ and 25°C./ PASS 10 mM citric acid, pH 6.0 7 days 11 0.5 mg/ml PHAD in 10 mMNa₂HPO₄, pH 6.0 25° C./ PASS 7 days

The data in the table above demonstrate that citrate, succinate, andphosphate buffers from about 10 mM to 50 mM are superior to certainothers buffers examined, and provide stability at the listedtemperatures for the listed time periods.

TABLE 4 Stability of PHAD at 25° C. for 30 or 60 days and 37° C. for 7,60 days, or 4 months in Select Buffers Condition/ Time Point Result 0.5mg/ml PHAD in 20 mM Na₂HPO₄ 25° C./ FAIL and 10 mM citric acid, pH 6.030 days 0.5 mg/ml PHAD in Phosphate buffered saline 25° C./ FAIL (PBS)30 days 0.5 mg/ml PHAD in 10 mM trisodium citrate, 25° C./ PASS pH 5.030 days 0.5 mg/ml PHAD in 10 mM trisodium citrate, 25° C./ FAIL pH 6.030 days 0.5 mg/ml PHAD in 10 mM Na₂HPO₄, 25° C./ FAIL pH 6.0 60 days 0.5mg/ml PHAD in 10 mM disodium succinate, 25° C./ FAIL pH 6.0 60 days 0.5mg/ml PHAD in 50 mM trisodium citrate, 25° C./ PASS pH 6.0 60 days 0.5mg/ml PHAD in 10 mM trisodium citrate, 37° C./ PASS pH 6.0 7 days 0.5mg/ml PHAD in 30 mM trisodium citrate, 37° C./ PASS pH 6.0 7 days 0.5mg/ml PHAD in 50 mM trisodium citrate, 37° C./ PASS pH 6.0 7 days 0.5mg/ml PHAD in 10 mM Na₂HPO₄, 37° C./ PASS pH 6.0 7 days 0.5 mg/ml PHADin 50 mM Na₂HPO₄, 37° C./ PASS pH 6.0 7 days 0.5 mg/ml PHAD in 10 mMtrisodium citrate, 37° C./ PASS 10 mM Na₂HPO₄, pH 6.0 7 days 0.5 mg/mlPHAD in 10 mM trisodium citrate, 37° C./ FAIL pH 6.0 60 days 0.5 mg/mlPHAD in 30 mM trisodium citrate, 37° C./ PASS pH 6.0 60 days 0.5 mg/mlPHAD in 50 mM trisodium citrate, 37° C./ PASS pH 6.0 60 days 0.5 mg/mlPHAD in 50 mM Na₂HPO₄, 37° C./ PASS pH 6.0 60 days 0.5 mg/ml PHAD in 10mM trisodium citrate, 37° C./ FAIL 10 mM Na₂HPO₄, pH 6.0 60 days 0.5mg/ml PHAD in 10 mM trisodium citrate, 37° C./ FAIL pH 6.0 4 months 0.5mg/ml PHAD in 30 mM trisodium citrate, 37° C./ PASS pH 6.0 4 months 0.5mg/ml PHAD in 50 mM trisodium citrate, 37° C./ PASS pH 6.0 4 months 0.5mg/ml PHAD in 50 mM Na₂HPO₄, 37° C./ PASS pH 6.0 4 months

The data in the table 4 demonstrate that citrate and phosphate buffersfrom about 30 mM to about 50 mM are superior to the other buffersexamined. The citrate and phosphate buffers provide stability at thelisted temperatures for the listed time periods. The data demonstratethe remarkable benefit of stability of increasing the citrate andphosphate concentrations to about 25 mM to about 50 mM, more preferably28 mM to about 50 mM, and most preferably 30 mM to about 50 mM. Thepreferred use of succinate as a buffer is shown from all of the datacollectively as described herein. Stability of PHAD at 37° C. for 60 ormore days in 30 mM to 50 mM citrate and phosphate buffers is unknown inthe prior art and is an important aspect of the invention.

TABLE 5 Stability of a 0.5 mg/ml PHAD Formulation of the Invention inSelect Buffers and Select Phosphatidylcholines at 25° C. for 60 dayswithout Polysorbate 80 and without Extrusion. The phosphatidylcholineswere prepared at about a molar ratio of 2.5 to 1 to PHAD. Condition/Time Point Result PHAD formulation with DPPC in 10 mM trisodium 25° C./PASS citrate, pH 6.0 60 days PHAD formulation with POPC in 10 mMtrisodium 25° C./ PASS citrate, pH 6.0 60 days PHAD formulation withDMPC in 10 mM trisodium 25° C./ PASS citrate, pH 6.0 60 days PHADformulation with DPPC in 50 mM trisodium 25° C./ PASS citrate, pH 6.0 60days PHAD formulation with DPPC in 10 mM disodium 25° C./ PASSsuccinate, pH 6.0 60 days PHAD formulation with POPC in 10 mM disodium25° C./ PASS succinate, pH 6.0 60 days PHAD formulation with DMPC in 10mM disodium 25° C./ PASS succinate, pH 6.0 60 days PHAD formulation withDPPC in 10 mM Na₂HPO₄, 25° C./ PASS pH 6.0 60 days

The data in the Table 5 demonstrate the remarkable and unexpectedbenefit of combining the citrate, succinate, and phosphate buffers atabout 10 mM to 50 mM with a phosphatidylcholine of the invention. Thecombination of the preferred buffers of the invention with aphosphatidylcholine results in superior stability of PHAD in theformulation compared to buffer alone.

TABLE 6 Condition/ Time Point Result 1 Adjuvant formulation of Example 1except 25° C./ FAIL reconstituted in WATER, not extruded, and 14 dayswithout polysorbate 80 2-8° C./ PASS 1 month 25° C./ FAIL 1 month 2-8°C./ PASS 2 months 25° C./ FAIL 2 months 2 Adjuvant formulation ofExample 1 (in 25° C./ PASS trisodium citrate, pH 6.0 as stated above) 7days not extruded and without polysorbate 80 25° C./ PASS 14 days 25°C./ PASS 1 month 25° C./ PASS 2 months 3 Adjuvant Formulation of Example1 2-8° C./ PASS (Lot 1214P69) 8 months 25° C./ PASS 8 months 4 AdjuvantFormulation of Example 1 2-8° C./ PASS (Lot ENG-1) 3 months 25° C./ PASS2 months 2-8° C./ PASS 5 months 25° C./ PASS 4 months 5 AdjuvantFormulation of Example 1 2-8° C./ PASS 3 months 25° C./ PASS 3 months2-8° C./ PASS 6 months 25° C./ PASS 6 months 6 FimCH Vaccine, 17 mMtrisodium citrate, 2-8° C./ PASS pH 5.4, consisting of: 0.1 mg/mladjuvant 4 days formulation of Example 1 without polysorbate 25° C./PASS 80 0.2 mg/ml FimCH, pH 5.4 4 days 2-8° C./ PASS 3 months 2-8° C./PASS 4 months

As shown in Table 6, it is the combination of citrate at about 10 mMwith specific molar ratios of DPPC to PHAD provides long-term stabilityat about 25° C.

As shown in the tables above, formulations prepared in water are stableshort-term when stored at 2° C. to 8° C.; however, the long sought-aftergoal is to reduce cold chain storage and management. The inventiveadjuvant formulation disclosed herein achieved this goal by providing anadjuvant formulation with extended stability at room temperature toabout 37° C. The data in Table 6 clearly show that the PHADconcentration of these formulations prepared in citrate buffer willremain stable for at least approximately 6 or more months at about 25°C. and potentially 2 to 3 years at 2° C. to 8° C.

The addition of citrate, succinate, or phosphate buffer to thePhosphatidylcholine:PHAD formulations remarkably and unexpectedlyenables storage at room temperature and exposure up to and at about 37°C. Phosphatidylcholine:PHAD formulations prepared in water can produceequivalent immunogenic responses in mice and rabbits but are not stableat about 25° C.

Example 12

Particle sizes and zeta potentials were determined on PHAD formulationsusing Dynamic Light Scattering with the Malvern Zetasizer® ZS90 orBrookhaven Instruments Corp. using ZetaPlus Particle Sizing software.The manufacturer's directions and recommendations were followed. Table 7provides representative data. Zeta potential values are used as onepiece of qualitative data estimating the electrical charge at a bilayerand as described herein. Stability of the PHAD formulations isexperimentally determined from particle size of the formulation andconcentration of PHAD.

TABLE 7 Mean Effective Zeta Diameter Potential Sample (nm) (mV) 1Adjuvant formulation of Example 1, not 207 −56 extruded and withoutpolysorbate 80 2 Adjuvant formulation of Example 1, 309 −37 containingDPPC:PHAD at a molar ratio of about 13:1, not extruded and withoutpolysorbate 80 3 Adjuvant formulation of Example 1, 293 −72 containingDMPC and not DPPC, not extruded and without polysorbate 80 4 Adjuvantformulation of Example 1, 202 −76 containing DMPC:DLPC and not DPPC, inan approximate molar ratio with PHAD at 1.2:1.2:1 (DMPC:DLPC:PHAD) notextruded and without polysorbate 80 5 Adjuvant formulation of Example 1without 79 −46 polysorbate 80 6 Adjuvant formulation of Example 1 71 −407 Adjuvant formulation of Example 1, 185 −73 extruded at roomtemperature and without polysorbate 80 8 DPPC alone (no PHAD) asprepared in 1393 −6 Example 1 without extrusion and without polysorbate80

As shown above, DPPC alone has a significantly less zeta potential andmuch larger mean particle size compared to the formulations of theinvention containing PHAD. The critical micelle concentration of DPPC isabout 0.46 nanomolar. These data offer evidence that DPPC alone is asignificantly different composition than a DPPC and PHAD composition.

Example 13

The particle sizes of PHAD formulations of Example 1 prepared with orwithout polysorbate 80 or glycerol were compared when stored at 2° C. to8° C. or about 25° C. to project (estimate) the stability of theparticle sizes of the PHAD formulations over 12 to 36 months. Multiplebatches of PHAD formulations were prepared and typically exhibitedparticles sizes between 70 to 100 nm immediately after extrusion. Thegoal for these PHAD formulations is to ensure that their mean effectivediameter remains preferably less than 150 nm over its shelflife, evenmore preferably less than 130 nm, which is predicted to be 2 to 3 yearsor longer based upon the data described herein. It is important to trendthe mean effective diameter over a certain time period atintermediate/accelerated conditions, e.g. 25° C., to assist withpredicting the particle size at 2° C. to 8° C. in approximately 2 ormore years.

Particle sizes and zeta potentials were determined using Dynamic LightScattering with the Malvern Zetasizer® ZS90 or Brookhaven InstrumentsCorp. using ZetaPlus Particle Sizing software. The instrumentmanufacturer's directions and recommendations were followed.

TABLE 8 Mean Time Effective Point/ Diameter Sample Condition (nm)Adjuvant formulation of Example 1 without 1 month/ 99 polysorbate 80 25°C. Adjuvant formulation of Example 1 with 1 month/ 104 0.1% polysorbate80 5° C. Adjuvant formulation of Example 1 1 month/ 79 2-8° C. Adjuvantformulation of Example 1 1 month/ 82 25° C. Adjuvant formulation ofExample 1 with 1 month/ 105 0.01% polysorbate 80 25° C. Adjuvantformulation of Example 1 without 4 months/ 83 polysorbate 80 2-8° C.Adjuvant formulation of Example 1 without 4 months/ 114 polysorbate 8025° C. Adjuvant formulation of Example 1 with 4 months/ 94 0.1%polysorbate 80 2-8° C. Adjuvant formulation of Example 1 with 4 months/106 0.1% polysorbate 80 25° C. Adjuvant formulation of Example 1 with 4months/ 90 0.01% polysorbate 80 2-8° C. Adjuvant formulation of Example1 with 4 months/ 114 0.01% polysorbate 80 25° C. Adjuvant formulation ofExample 1 4 months/ 86 25° C. Adjuvant formulation of Example 1 5months/ 82 2-8° C. Adjuvant formulation of Example 1 6 months/ 101 25°C. Adjuvant formulation of Example 1 6 months/ 94 2-8° C. Adjuvantformulation of Example 1 without 8 months/ 117 polysorbate 80 2-8° C.

The data presented in Table 8 in this example project that the particlesizes of the PHAD formulations stored at 2° C. to 8° C. withoutpolysorbate 80 will remain below 150 nm for approximately a minimum of 2years, and suggest potentially 3 years as described below. In thisspecific example and only for the purpose of this test, stable meansthat the mean effective diameter remains below 150 nm within thelimitations of the instrument. To those skilled in the art, data from25° C. is an intermediate/accelerated condition used to project a shelflife at the long term storage condition of 2° C. to 8° C. These dataclearly project that the particle sizes of these PHAD formulations willremain stable for at least 6 or more months at about 25° C. andpotentially 3 years at 2° C. to 8° C. As described herein, thisstability of these PHAD formulations at 25° C. is unknown in the priorart and unexpected.

Example 14

A Comparison of the Formulations of the Invention to Other AdjuvantFormulations at Inducing anti-FimH Antibodies in Mice.

The PHAD formulation of the invention or aqueous formulation (Aqueousformulation in this example refers to the molar ratio of lipid toadjuvant described in U.S. Pat. No. 6,491,919 and US Patent Application20080131466) used below in this example were prepared as described inExample 1 with the following exceptions. The specified lipid(s) and/ormolar ratios (shown in parentheses) were used and sonication occurred atabout 45° C. for about 30 minutes to 2 hours as needed to achieve ahomogenous suspension. All lipids were purchased from Avanti PolarLipids as previously described herein.

Female C3H/HeN mice (approximately 9 weeks old) were purchased fromCharles River laboratories (Wilmington, Mass.). Mice were injected viathe intramuscular route (IM) in the right thigh under light isofluraneanesthesia (Henry Schein, Melville, N.Y.) in a 50 μl volume using a 30gauge needle. Mice were vaccinated with 12.5 μg PHAD or Freund'sadjuvant C (subcutaneous administration), and 15 μg FimCH, on days 1 and29. As known to those skilled in the art, when used, complete Freund'sadjuvant was given on day 1 and incomplete Freund's adjuvant on day 29.

ELISA for serum antibody detection: Sera was collected from the mice atsacrifice and analyzed by ELISA for anti-FimH antibodies. FimH truncateT3 was adhered to Immulon 4 HBX plates (ThermoFisher) at 2 μg/ml in PBSovernight at 4° C. After washing with PBS+0.05% Tween 20, open bindingsites were blocked with 1.5% BSA (Sigma Aldrich) in PBS for 1 hr. Afterwashing, dilutions of sample sera (in PBS with 0.05% Tween 20, 0.1% BSA,0.5% methyl α-D-mannopyranoside) were incubated for 2 hrs. Afterwashing, 1:500 diluted biotinylated Goat anti-Mouse IgG detectionantisera (Sigma Aldrich) in sample dilution buffer was incubated in thewells overnight at 4° C. After washing, 1:25,000 diluted Avidin-HorseRadish Peroxidase (HRP, Sigma Aldrich) in sample dilution buffer wasincubated in the wells for 20 minutes. After washing, HRP activity wasdetected using TMB substrate in Phosphocitrate buffer (Sigma Aldrich).Optical density was read at 630 nm using a VersaMaxPLUS microplatereader and analyzed using SoftMax Pro software (Molecular Devices,Sunnyvale, Calif.). Antibody titer was defined as the highest dilutionwith signal above background.

TABLE 9 Antibody Dilution Adjuvant/Formulation Titers No Adjuvant; N = 6mice 1:20,000  Experiment 1: Reference Adjuvant:Freund's adjuvant; N = 61:200,000 mice Experiment 2: Reference Adjuvant:Freund's adjuvant; N = 71:200,000 mice Experiment 1: DPPC:PHAD (1:3.9) aqueous formulation;1:200,000 N = 8 mice Experiment 2: DPPC:PHAD (1:3.9) aqueousformulation; 1:200,000 N = 10 mice DPPC:PHAD (2.6:1) adjuvantformulation; N = 10 mice 1:400,000 DPPC:PHAD (13:1) adjuvantformulation; N = 10 mice 1:400,000 DPPC:DPPG:PHAD (2.6:0.3:1) adjuvantformulation; N = 10 1:400,000 mice DPPC:DPPG:PHAD (2.6:2.6:1) adjuvantformulation ; N = 10 1:400,000 mice

As clearly shown in Table 9, a molar ratio of DPPC to PHAD of about 2:1to about 13:1 was superior at enhancing an immune response to FimH inmice when compared to no adjuvant, Freund's adjuvant, or the aqueousformulation of DPPC to PHAD at a molar ratio of 1:3.9. Freund's adjuvantis considered a standard adjuvant used in animal experiments. The datashows that the molar ratio of the invention described herein of about2:1 to 13:1 is superior to this frequently used preclinical adjuvant.This is one aspect of the invention that demonstrates its superiority tothe prior art formulations. The data in Table 9 also demonstrate thatadding DPPG into these formulations does not diminish the intended useof the PHAD formulations, i.e., to enhance an immune response to FimH.

Example 15

Determination that the Formulations of the Invention remain a HomogenousMixture within 24 Hours. The procedure of Example 1 was used to preparethe PHAD formulation with a molar ratio of DPPC:PHAD at about 2.5 to 1,except polysorbate 80 was not added. The vial containing the PHADformulation was gently inverted about three to five times. Then, thePHAD formulation was allowed to remain at room temperature for about 24hours. After 24 hours and without inverting, shaking, or stirring thePHAD formulation, small aliquots were carefully removed from the top,middle, and bottom of the PHAD suspension. These aliquots were analyzedfor PHAD concentration via HPLC as previously described herein. Theresults demonstrated that the aliquots from the top, middle, and bottomcontained equivalent PHAD concentrations. These results demonstrate thePHAD formulation of the invention does not settle within 24 hours.

Example 16 Formulations of the Invention Used in Human Clinical Study

The adjuvant formulation of Example 1 and FimCH were prepared under cGMPfor use in a human clinical study involving females of about 21 to 64years of age. A vaccine of FimCH and PHAD formulation was prepared byadding a predetermined volume of a PHAD formulation to a vial of FimCHas previously described herein to obtain the preferred concentrations ofFimCH and PHAD per each vial. An appropriate volume of the preparedvaccine was injected IM (intramuscular) to administer either 50 μg orabout 107 μg of FimCH with either 10 μg, 20 μg, or about 40 μg of PHADto each female subject.

From these IM injections in humans, the vaccine demonstrates that theadjuvant formulation of the invention with FimCH produces less severeinjection site and systemic reactions in humans compared to certainother known adjuvant formulations used in humans. This is a remarkableaspect of the invention. Interim Data from Injection Site and SystemicReactions are shown below for number of injections as of the interimanalysis per female. The human study is ongoing and the females in thestudy are scheduled to receive 4 injections.

TABLE 10 FimCH/PHAD Severe Injection Number of dose in Number of Siteand Females micrograms Injections Systemic Reactions Group 1 5 107 μg/0μg  3 to 4 None Group 2 8 50 μg/10 μg 2 to 3 None Group 3 16 50 μg/20 μg1 to 2 None Group 4 8 50 μg/40 μg 1 None

At the time of this interim analysis, more than 60 IM injections to 37females had been made and no severe injection site and systemicreactions have been observed. At the time of this interim analysis,females in Group 2 have demonstrated an antibody response to FimH aftertwo IM injections greater than 10-fold from initial values prior tovaccination. These data demonstrate the vaccine is producing theintended antibody response against FimH. Females in this study willreceive up to 4 IM injections of the vaccine. Groups 5 and 6 will openenrollment for females with a history of recurrent UTI.

In comparison, administration of the Cervarix vaccine that contains theadjuvants MPL with alum results in approximately 8% to more than 10% ofsubjects exhibiting severe injection site reactions and systemicreactions. As reported in the publication of Treanor et al. (Vaccine,2013, 31(48), 5760-5), PHAD has been prepared in different formulationswith squalene and administered to humans. Per this report, 5 μg of PHADin this formulation resulted in severe local reactions, chills, andrigor and was so limiting that use of this formulation in future studiesonly contained 2 or less micrograms of PHAD. At 1 micrograms of PHAD inthis formulation, severe injection site reactions and systemic reactionswere still observed. In this formulation of Treanor et al. at 2 or lessmicrograms of PHAD, the benefits of the adjuvant PHAD producing animmune response are limited thereby robbing the potential of PHAD due toan inferior formulation. However, the adjuvant formulation of theinvention overcomes this limitation with superior formulations thatenable administration of up to 50 μg of PHAD, or potentially more, withless severe injection site and systemic reactions. These human dataoffer evidence that PHAD can be administered to humans at 100 μg or moremicrograms with the formulations described herein. The adjuvantformulations of the invention are superior to those formulationspreviously attempted.

All references, including without limitation all papers, publications,presentations, texts, reports, manuscripts, brochures, internetpostings, journal articles, periodicals, and the like, cited in thisspecification are hereby incorporated by reference. The discussion ofthe references herein is intended merely to summarize the assertionsmade by their authors and no admission is made that any referenceconstitutes prior art. The inventors reserve the right to challenge theaccuracy and pertinence of the cited references.

It is intended that all patentable subject matter disclosed herein beclaimed and that no such patentable subject matter be dedicated to thepublic. Thus, it is intended that the claims be read broadly in light ofthat intent. In addition, unless it is otherwise clear to the contraryfrom the context, it is intended that all references to “a” and “an” andsubsequent corresponding references to “the” referring back to theantecedent basis denoted by “a” or “an” are to be read broadly in thesense of “at least one.” Similarly, unless it is otherwise clear to thecontrary from the context, the word “or,” when used with respect toalternative named elements is intended to be read broadly to mean, inthe alternative, any one of the named elements, any subset of the namedelements or all of the named elements.

In view of the above, it will be seen that the several advantages of theinvention are achieved and other advantageous results obtained. Itshould be understood that the aforementioned embodiments are forexemplary purposes only and are merely illustrative of the many possiblespecific embodiments that can represent applications of the principlesof the invention. Thus, as various changes could be made in the abovemethods and compositions without departing from the scope of theinvention, it is intended that all matter contained in the abovedescription as shown in the accompanying drawings shall be interpretedas illustrative and not in a limiting sense.

Moreover, one of ordinary skill in the art can make various changes andmodifications to the invention to adapt it to various usages andconditions, including those not specifically laid out herein, withoutdeparting from the spirit and scope of this invention. Accordingly,those changes and modifications are properly, equitably, and intended tobe, within the full range of equivalents of the invention disclosed anddescribed herein.

1. A method for administering a vaccine composition to a humancomprising the steps of: administering to the human a vaccinecomposition comprising an adjuvant formulation comprising about 20 μg toabout 50 μg of phosphorylated hexaacyl disaccharide (PHAD), aphosphatidylcholine, and a buffer selected from the group consisting ofcitrate and succinate from about 10 mM to about 50 mM, wherein thephosphatidylcholine is at a molar ratio with PHAD at about 1:1 to about20:1, and the vaccine composition is an aqueous buffered suspension. 2.(canceled)
 3. (canceled)
 4. The method of claim 1, wherein the PHAD ispresent in an amount from about 40 μg to about 50 μg.
 5. The method ofclaim 1, wherein the buffer is at a concentration of 15 mM to about 50mM.
 6. The method of claim 1, wherein the buffer is at a concentrationof 20 mM to about 50 mM.
 7. The method of claim 1, wherein PHAD iscomprised of


8. The method of claim 1, wherein the buffer is citrate.
 9. The methodof claim 1, wherein the buffer is succinate.
 10. The method of claim 1,wherein the vaccine composition comprises an effective amount of FimCHor truncated FimH.
 11. The method of claim 1, wherein the vaccinecomposition comprises an effective amount of FimCH.
 12. The method ofclaim 1, wherein the vaccine composition is for the treatment ofrecurrent urinary tract infections.
 13. The method of claim 1, whereinphosphatidylcholine is selected from the group consisting of1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC),1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC),1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC),1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), and1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC).
 14. The method of claim1, wherein the phosphatidylcholine is selected from the group consistingof DMPC, DPPC, and DSPC.
 15. The method of claim 1, wherein thephosphatidylcholine is at a molar ratio with PHAD at about 2:1 to about5:1.
 16. The method of claim 13, wherein the phosphatidylcholine is at amolar ratio with PHAD at about 2:1 to about 5:1.
 17. The method of claim14, wherein the phosphatidylcholine is at a molar ratio with PHAD atabout 2:1 to about 5:1.
 18. The method of claim 1, wherein the vaccinecomposition has a mean particle size of 150 nanometers or less.
 19. Themethod of claim 1, wherein the vaccine composition is essentially freeof a second adjuvant.
 20. The method of claim 1, wherein the vaccinecomposition is substantially free of saline.
 21. The method of claim 20,wherein the PHAD is comprised of


22. The method of claim 1, wherein the vaccine composition issubstantially free of cholesterol.
 23. The method of claim 1, whereinthe vaccine composition is essentially free of cholesterol.
 24. A methodfor administering a vaccine composition to a human comprising the stepsof: administering to the human a vaccine composition comprising anadjuvant formulation comprising about 20 μg to about 50 μg ofphosphorylated hexaacyl disaccharide (PHAD); phosphatidylcholine at amolar ratio with PHAD at about 2:1 to about 5:1; phosphatidylcholine isselected from the group consisting of DMPC, DPPC, DSPC, POPC, and DOPC;and a buffer selected from the group consisting of citrate and succinatefrom 15 mM to about 50 mM, wherein the vaccine composition is an aqueousbuffered suspension.
 25. The method of claim 24, wherein the PHAD iscomprised of


26. The method of claim 24, wherein the buffer concentration is from 25mM to about 50 mM.
 27. The method of claim 24, wherein the vaccinecomposition is essentially free of a second adjuvant.
 28. The method ofclaim 24, wherein the vaccine composition comprises an effective amountof FimCH or truncated FimH.
 29. The method of claim 24, wherein thevaccine composition comprises an effective amount of FimCH.
 30. Themethod of claim 24, wherein the vaccine composition comprises about 40μg to about 50 μg of PHAD, the buffer is citrate, and an effectiveamount of FimCH, wherein the vaccine composition is essentially free ofa second adjuvant.
 31. An adjuvant formulation for a vaccine comprising:phosphorylated hexaacyl disaccharide (PHAD); a phosphatidylcholine; and,a buffer selected from the group consisting of citrate and succinate,wherein the buffer concentration is from about 10 mM to about 50 mM, andthe phosphatidylcholine is at a molar ratio with PHAD at about 1:1 toabout 20:1.
 32. The adjuvant formulation of claim 31 wherein the he PHADis comprised of